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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCX 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 : 
A61K 38/17, C12N 5/00 



A2 



(11) International Publication Number: WO 99/45949 

(43) International Publication Date: 16 September 1999 (16.09.99) 



(21) International Application Number: PCT/US99/04003 

(22) International Filing Date: 24 February 1999 (24.02.99) 



(30) Priority Data; 

09/037 J 18 



9 March 1998 (09.03.98) 



US 



(71) Applicant: GENETICS INSTITUTE. INC. [US/US]; 87 Cam- 

bridgePark Drive, Cambridge. MA 02140 (US). 

(72) Inventors: WOOD. Clive. R.; 2 Hawthorne Place #17R, 

Boston. MA 02114 (US). FTTZ. Lori. Jo; 13 Palmer Street, 
Arlington, MA 02174 (US). 

(74) Agent: LAZAR, Steven, R.; American Home Products Corpo- 
ration. Legal Affairs, Patent and Trademark Dept.-2B, One 
Campus Drive, Atm.: Kay E. Brady, Parsippany. NJ 07054 
(US). 



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



Published 

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



(54) TiUe: USE OF FOLLISTATIN TO MODULATE GDF-8 AND BMP-1 1 



(57) Abstract 



Methods are provided for the modulation of the effects of GDF-8 and BMP-1 1, particulariy on neural and muscular disorders 
administration of follistatin for treating neural, muscle, disorders which are characterized by an abnormality in the levels or activity of 
GDF-8 or BMP-1 1. 



FOR THE PURPOSES OF INFORMATION ONLY 
Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT. 



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Annenia 


FI 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of MoWova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


'Hnlancnistan 


BF 


Builcina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


EG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


UZ 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CG 


Congo 


KE 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia. 


CH 


Switzerland 


KG 


Kyigyzstan 


NO 


Norway 


ZW 


Zimbabwe 


CI 


C6te d'lvoirr 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






cu 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






cz 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


U 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


S£ 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







wo 99/45949 



PCTAJS99/04003 



USE OF FOLLISTATIN TO MODULATE GDF-8 AND BMP-1 1 

FIELD OF THE INVENTION 
The present invention relates to use of follistatin to modulate the activity of a 
growth and differentiation factor [GDF] known as GDF-8. More particularly, the 
invention relates to use of follistatin for treating neural and muscle, disorders which are 
10 related to modulation of the levels or activity of GDF-8 or closely related factors, 
including bone morphogenetic protein- 1 1 [BMP- 1 1 ], also known as GDF- 1 1 . 
BACKGROUND OF THE INVENTION 
Bone morphogenetic proteins (BMPs) and growth/differentiation factors (GDFs) 
are part of a family of proteins which have been identified as having the ability to 
15 induce the growth, formation, differentiation and maintenance of various tissues, 
including bone, cartilage, tendon/ligament, muscle, neural, and various organs. BMPs 
and GDFs are subfamilies within the TGF-p superfamily. 

The TGF-p superfamily of proteins have been shown to bind to serine/threonine 
kinase receptors. Massague, Cca, 69:1067-1070 (1992); Attisano et al.. Cell 68:97-108 
20 (1992); Lin et al.. Cell, 68:775-785 (1992); Wang et al„ Cell 67:797-805 (1991). 

Similarly, activin receptors have been isolated and characterized as a predicted 
transmembrane serine kinase. Mathews et al., Cell 65:973-982 (1991); Nakamura et 
al., J. Biol. Chem. 267:18924-18928 (1992). Ebner et al.. Science , 260:1344-1348 
(1993) describe the existence of Type I and Type II TGF-p receptors, and the effects of 
25 the Type I receptor on binding of TGF-p to the Type n receptor, 

Follistatin is a protein which has been identified as a molecule which is able to 
bind to activin, another member of the TGF-p superfamily, and as a possible antagonist 
of activin. United States Patent 5,545,616. Accordingly, follistatin has been suggested 
for possible use to predict and/or prevent preterm labor and to suppress FSH secretion 
30 from the pituitary [US Patent 5,545,616]; to have inhibin like activity [United States 
Patent 5,041,538]; and for use in rheumatoid arthritis [AU9675056, Kaneka Corp] 



wo 99/45949 PCT/US99/04003 

5 SUMMARY OF THE INViENTION 

Accordingly, the present invention provides methods for modulating the effects 
on cells of a protein selected from the group consisting of growth and differentiation 
factor 8 [GDF-8] and bone morphogenetic protein 11 [BMP-11], said method 
comprising administering to said cells an effective amoimt of foUistatin. The invention 

10 further provides methods for blocking the effects on cells of GDF-8 or BMP-1 1 and 
methods for treating a disorder associated with neural or muscular effects of GDF-8 or 
BMP-1 1, said method comprising administering to said cells an effective amount of 
foUistatin. 

In one embodiment, the present invention comprises methods of modulating the 

15 production and/or activity of vGDF-8 or BMP-11, thereby affecting the growth, 
formation, differentiation and maintenance of cells using a foUistatin protein, or a DNA 
molecule encoding a foUistatin protein. The present invention further comprises 
treatment of disorders which are associated with the production, metabolism and 
activity of GDF-8 or BMP-1 1 . Preferred embodiments include u-eatment of diseases 

20 and disorders involving neural or neuronal and muscle ceUs and tissue. These disorders 
include neurodegenerative and musculodegenerative diseases, such as muscle or nerve 
wasting, muscle or nerve atrophy, amyotrophic lateral sclerosis, Alzheimer's Disease, 
Parkinson's Disease and muscular dystrophy. The present invention further includes 
the use of foUistatin for the treatment of traumatic or chronic injury to the spinal cord, 

25 or to the nerve or muscle system. 

DFT ATT Fn DESCRIPTION OF THE INVENTION 
TGF-P protein, such as BMPs and GDFs, are characterized by their ability to 
promote, stimulate or otherwise induce the growth, formation, differentiation and 
maintenance of various tissues, including bone, cartilage, tendon/ligament, muscle, 

30 neural, and various organs. GDF-8 has been shown to exhibit particular activity on 
muscle, adipocyte and neural tissue. BMP-1 1 has been shown to exhibit activity on 
neural^cells, particularly on neuronal cells. 

Two forms of foUistatin (FS) are produced as a result of alternative splicing. 
These forms are FS-288 and FS-315. The FS-315 form has also be shown to be 

35 proteolytically processed to form FS-303 (Sugino et al., J.Biol. Chem . 



2 



wo 99/45949 PCT/US99/04003 
5 268: 15579(1993)). Recombinant forais of each of these molecules are expected to have 
different properties (Sumitomo et al., Biochem. Biophvs. Acta 208:10995)) and are 
envisaged to be useful for inhibiting the action of GDF-8 and BMP-1 1 . 

The expected properties of follistatin, in light of the present showing, include 
differential ability to interact with cell surfaces ,and bind heparin and heparan sulphate 

10 proteoglycans (Nakamura et al., J. Biol. Chem. 266:19432 (1991); Sumitomo et al., 
Biochem Biophvs. Acta 208:1 (1995)). These properties maybe suboptimal in the FS 
used for therapeutic use. As a consequence, site-directed mutagenesis may be used to 
alter this property. Specifically, this can involve changing or deleting the basic residues 
responsible for heparin binding, at residues 72-86 (Inouye et al., Mol Cell; Endocrinol. 

15 90:1(1992)). 

Follistatin is useful, among other uses, for the identification of BMPs, the 
identification of further BMP receptors, and the identification of ligands or molecules, 
including antibodies, which are able to mimic the binding characteristics of BMPs. 
These ligands may act as agonists or antagonists, depending upon the individual ligand. 

20 The ability of follistatin to block or modulate the activity of GDF-8 and BMP- 1 1 may 
be characterized in an assay for BMP activity, such as the animal cap assay, described 
at Example 2 below. The follistatin molecules are also useful in inhibiting the effects 
of GDF-8 and BMP-1 1, where such inhibition is desired. 

Because of the known activities of GDF-8 and BMP- 1 1 , the present invention 

25 will find use in treating muscle-related disorders, diseases of the nervous system 
(including infections), vascular disorders,, trauma, metabolic derangements, 
demyelinating diseases (including multiple sclerosis), neuronal diseases (including 
Alzheimer's disease , Parkinson's disease and Huntington's chorea; and including motor 
neuron diseases such as amyotrophic lateral sclerosis, primary lateral sclerosis and 

30 Werdnig-Hofftnann disease), epilepsy, syringomyelia, peripheral neuropathy, congenital 
anomalies and tumors. Muscle-relatcd conditions for treatment include without 
limitation muscular dystrophies (such as severe and benign X-linked muscular 
dystrophy, limb-girdle dystrophy, facioscapulohumeral dystrophy, myotinic dystrophy, 
distal muscular dystrophy, progressive dystrophic ophthalmoplegia, oculopharyngeal 

35 dystrophy and Fukuyama-type congenital muscular dystrophy), congenital myopathy, 



3 



wo 99/45949 PCT/US99/04003 
5 myotonia congenital, familial periodic paralysis, paroxysmal myoglobinuria, myasthenia 
gravis, Eaton-Lambert syndrome, secondary myasthenia, denervation atrophy, 

FoUistatin proteins useful in the present invention include human foUistatin, 
disclosed in Shimasaki et al., PNAS:USA 85:4218-4222 (1988); porcine foUistatin, 
disclosed in Ueno et al., PNAS:USA 84:8282-8286 (1987); and bovine foUistatin, 

10 disclosed in Robertson et al,, Biochem. Biophvs. Res. Conmiun. 149:744-749 (1987). 
The disclosures of each of these publications is hereby incoiporated by reference herein. 
In addition, truncated polypeptides which comprise partial fragments of the full 
foUistatin polypeptides, and which retain the ability to bind to GDF-8 and BMP-1 1, may 
also be useful for the present invention. In particular, functional fragments of foUistatin 

15 sequences, which maintain the abihty to modulate, block or otherwise affect GDF-8 
and/or BMP-1 1 activity, are useful for the methods of the present invention. The 
identification of a partial foUistatin polypeptide as a functional fragment of foUistatin 
may readily be determined, for example, using the assay described in Example 2. 

The present invention also includes fusions of foUistatin with other molecules. 

20 This includes the fusion of FS-288, FS-3 1 5 or FS-303 sequences with the hinge, CH2 
and CHS domains of a human immunoglobulin gamma isotype, e.g., gamma 1 or 4. 
Such a fusion protein is expected to produce a dimeric molecule, with the improved 
pharmacokinetics expected for an immunoglobulin Fc fusion. In addition, the constant 
domains or secretory tailpieces of alpha or mu iiiununoglobulin heavy chains may be 

25 fused to FS in order to generate polymeric forms of FS, 

The component portion of FS responsible for interacting with GDF-8 and BMP- 
1 1 can be identified and used to generate functional fragments of FS, fusion proteins, 
or as the basis for other therapeutic utiUties. The human FS ^ene contains fovu* domains 
each encoded on a separate exon, in addition to an exon encoding a N-terminal signal 

30 sequence, and an exon encoding the C-terminal extension that results in FS-3 15 
(Shhnasaki et al. . Proc. Natl. Acad. Sci USA 85:4218(1995)). The regions responsible 
for GDF-8 and/or BMP-1 1 binding can be determined and prepared by the methods 
described in Example 3. 

For use in the methods of the present invention, the purified foUistatin proteins 

35 and functional fragments thereof may be produced through purification from native 



4 



wo 99/45949 PCTAJS99/04003 

5 tissues, or recombinantly by culturing a host cell transformed with a DNA sequence 
comprising the DNA coding sequence described in any of the above publications. In 
addition to the native DNA coding sequences, coding sequences which can be used 
include sequences which code for the above, but which differ in codon sequence due 
to the degeneracies of the genetic code or allelic variations (naturally-occurring base 

10 changes in the species population which may or may not result in an amino acid 
change), as well as'DNA sequences which hybridize under stringent hybridization 
conditions [see, T. Maniatis et al. Molecular Cloning f A Laboratorv Manual) . Cold 
Spring Harbor Laboratory (1982), pages 387 to 389] to the DNA sequences described 
in the above publications and encode a protein having the ability to bind to GDF-8 or 

15 BMP-1 1. Variations in the DNA sequences disclosed in the above publications which 
are caused by point mutations or by induced modifications (including insertion, 
deletion, and substitution) to enhance the activity, half-life or production of the 
follistatin polypeptides encoded thereby are also useful for the present invention. 

The present invention may include gene therapy, in which transfection of cells 

20 with DNA molecules encoding follistatin or functional fragments thereof is made in 
order to achieve binding of the follistatin to GDF-8 and/or BMP- 1 1 present within the 
transfected cells or in the environment of the transfected cells, and thereby modulate or 
block the effects of GDF-8 and/or BMP-1 1 on those cells. For example, cells which 
express the follistatin proteins may reduce or eliminate the effects of an excess of GDF- 

25 8 or BMP-1 1 in an organism or cell. The increased follistatin may be desirable for 
minimizing negative effects of GDF-8 or BMP- 1 1 . or may act as a complex with GDF-8 
or BMP-1 1 to enhance or increase activity. 

Follistatin proteins or functional fragments thereof may also be useful in a 
process for isolating GDF-8 or BMP-1 1 in a purification process. In such a process, 

30 follistatin may be incorporated into a colunm or a resin which may be used for the 
commercial production of GDF-8 or BMP-1 1 from tissue samples or via recombinant 
processes. The follistatin or functional fragments thereof are used to bind to the GDF-8 
or BMP-1 1, and later subjected to conditions which r-esult in the release of said bound 
protein. 



5 



wo 99/45949 PCTAJS99/04003 

The present invention includes therapeutic methods comprising administering 
a foUistatin containing composition topically, systematically, or locaUy as an implant 
or device. When administered, the therapeutic composition for use in this invention is 
preferably in a pyrogen-free, physiologically acceptable form. Further, the composition 
may desirably be encapsulated or injected in a viscous form for delivery to the desired 
site. Therapeutically useful agents, such as growth factors (e.g., BMPs, TGF-p, FGF, 
IGF), cytokines (e.g., interleukins and CSFs) and antibiotics, may also optionally be 
included in or administered simultaneously or sequentially with,; the FoUistatin 
composition in the methods of the invention. 

There is a wide range of methods which can be used to deliver the cells 
expressing foUistatin proteins to a site for use in modulating a GDF-8 or BMP- 11 
response. In one embodiment of the invention, the cells expressing foUistatin protein 
can be delivered by direct application, for example, direct injection of a sample of such 
cells into the site of tissue damage. In a particular embodiment, these cells can be 
purified. In a preferred embodiment, the cells expressing foUistatin protein can be 
delivered in a medium or matrix which partially impedes their mobility so as to localize 
the cells to a site of injury. Such a medium or matrix could be semi-solid, such as a 
paste or gel, including a gel-like polymer. Alternatively, the medium or matrix could 
be in the form of a solid, preferably, a porous solid which will allow the migration of 
ceUs into the solid matrix, and hold them there while allowing proUferation of the ceUs. 

In a method of the present invention, the cells expressing foUistatin are applied 
in the desired site as described above, and GDF-8 or BMP-1 1 is applied. The factor 
may be applied simultaneously or immediately following application of the cells 
expressing foUistatin. The BMP may be apphed in manners known in the art, such as 
described in the above patents, as weU as in United States Patent 5,171,579, the 
disclosure of which is also hereby incorporated by reference. 
Expression of FoUistatin Protein 

In order to produce foUistatin protein, the DNA encoding the desired protein is 
transferred into an ^propriate expression vector and introduced into manmialian -cells 
or other preferred eukaryotic or prokaryotic hosts by conventional genetic engineering 



wo W4S949 PCT/US99/04003 

5 lechniques. The presently preferred expression system for biologically active 
recombinant follistatin protein is stably transformed mammalian cells. 

The following examples detail presently preferred embodiments of the present 
invention. Numerous modifications and variations in practice thereof are expected to 
occur tOvthose skilled in the art upon consideration of these descriptions. Those 

10 modifications and variations are believed to be encompassed within the claims 
appended hereto. The examples do not in any way limit the invention. 

EXAMPLES 

EXAMPLE 1. BIAcore binding assay: 

15 Purified follistatin was coupled to a carboxymethyl dextran layer of a CMS 

research grade chip on a Biacore 2000 instrument using standard amine coupling 
procedures according to the manufacturer's instructions. The buffer used for 
immobilization was 10 mM sodium acetate pH 4. Typically about 7,000 response units 
(kU) of follistatin were inmiobilized by this procedure. Purified BMP and GDF 

20 proteins were each injected over the immobilized follistatin for 10 minutes at 2 pl/min. 
The running buffer used for screening was 10 mM sodium phosphate pH 7.4, 300 mM 
sodium chloride, 3.4 mM ethylenediaminetetra-acetic acid, 0.005% (v/v) Tween 20 and 
the temperamre was maintained at 22 ''C. Binding was quantified as an increase in RU 
at 60 sec after the end of the injection compared to a baseline established 20 sec prior 

25 to injection. Specific binding was shown by coinjection of soluble follistatin and the 
BMP-1 1 andGDF-8 proteins. 
Results: 

Results from the Biacore screen showed that both GDF-8 and BMP-1 1 bound 
follistatin. This binding was comparable to the positive control, activin. The binding 
30 was specific, as demonstrated by the fact that no binding was observed when GDF-8 or 
BMP-1 1 was preincubated and coinjected with excess soluble follistatin. 



7 



wo 99/45949 PCTAJS99/04003 

EXAMPLE 2: Animal Cap Assay Method 

The Xenopus animal cap assay has been used to assess the biological activity of 
BMP proteins, Xenopus eggs were fertilized in vitro and allowed to develop until the 
blastula stage. The ectodermal or animal cap of the embryo was excised and cultured 
in media containing the protein of interest for 5-6 hours. The explants were then 
transferred to fresh media without protein. The animal caps were cuhured ovemight 
and the activity of the protein was evaluated the next day by morphology, histology, and 
RT-PCR using molecular markers of mesoderm, neural tissue, and endoderm. 
Animal Cap Assay Results 

Both GDF-8 and BMP-1 1 caused animal caps to elongate and induced dorsal mesodenn 
(muscle) and neural tissue at doses (50ng/ml) comparable to that for factors that have 
been shown previously to induce these tissues (e.g., activin). Follistatin was able to 
inhibit the ability of both GDF-8 and BMP-1 1 to induce elongation and mesodermal 
tissue in animal caps. GDF-8 was blocked by a 5 fold excess of follistatin (lOOng/ml 
GDF-8 and 500ng/ml follistatin) while BMP-1 1 was blocked by a 10 fold excess of 
follistatin (BMP-1 1 5Qng/ml and 50Qng/ml follistatin). Together, the Biacore binding 
results and inhibition on the Xenopus animal cap assay demonstrate that follistatin is 
an anugonist of GDF-8 and BMP-1 1, and is able to modulate the activity of these two 
factors. 

EXAMPLE 3: Determination of Functional Fragments of Follistatin 

Functional fragments of Follistatin, and the components of Follistatin that are 
necessary for the preparation thereof, are defined by generating a series of FS mutants 
each with an additional exon deleted from the 3' end. The six exons of FS are 
numbered i to 6. The mutants will consist of exons 1-5, 1-4, 1-3 and 1-2 and the 
binding of each form will be compared with wild-type FS (1-6). This will identify the 
domain or domains responsible for ligand binding. Specific residues that are critical for 
binding to ligand will then be identified using site-directed mutagenesis. 

The 1-5, 1-4, 1-3 and 1-2 forms will be generated by using oligonucleotide 
primers and the polymerase chain reaction (PGR). The template for this amplification 
will be the FS cDNA, either from a plasmid clone or as the result of random hexamer- 
primed first strand cDNA synthesis from primary tissue poly A+ RNA (eg., from ovary 



8 



wo 99/45949 PCTAJS99/04003 

5 RNA). A forward (5') primer based on the start codon of FS will be used in each 
amplification, and combined with a reverse (3') primer that anneals to the 3* coding 
sequence of the final exon {e.g., exon 5 for the 1-5 form) and introduces a stop codon 
immediately after the final exon. Recognition sequences of restriction endonucleases 
will also be added to the 5' end of each primer to facilitate molecular cloning of the 
10 PCR product into an expression vector. PCR conditions and components will be chosen 
to minimize the introduction of point mutations, and the resulting clones will be 
analyzed by nucleotide sequencing to ensure the correct FS sequence is present in each 
construct. 

The forward primer is called FS-forward. The reverse primer for generating 1 -5 

15 is called FS-reverse 5; for 1-4 is called FS-reverse 4; for 1-3 is called FS-reverse 3 ;and 
for 1-2 is called FS-reverse 2. Potential sequences for these primers are given below. 
The FS sequences responsible for interacting with GDF-8, BMP-1 1 and activin may be 
identical. If the binding sites are discrete or overlapping, mutagenesis can be used to 
abolish binding to specific FS ligands. This can be achieved by alanine-scanning 

20 mutagenesis and testing of each mutant for binding to each of the three ligands. 
FS-forward: 5'-dCCAGGATGGTCCGGGCGAGG-3' [SEQIDNO:!] 
FS-reverse 5: 5'-dTCAGTTGCAAGATCCGGAGT-3' ISEQ ID N0:2] 
FS-reverse 4: 5'-dTCATTTGATACACnTCCCTCAT-3' [SEQIDNO:3] 
FS-reverse 3: 5^dTCACTTTTTACATCTGCCTTGGT-3' [SEQ ID NO:4] 

25 FS-reverse 2: 5'-dTCATTCnTACAGGGGATGCAGT-3' [SEQIDNO:5] 

Using techniques and primers similar to those described above, a series of FS 
mutants each with an additional exon deleted from the '5' end is generated in order to 
determine whether the N-terminal portion of the FoUistatin protein are required for 
functional fi-agments of FoUistatin, These mutants will consist of exons 3-6, 4-6, 5-6 

30 and 6, and the binding of each form will also be compared with wild-type FS (1-6). The 
first exon, including the signal sequence, will be included on each constmct to facilitate 
the proper secretion of each molecule. 



9 



wo 99/45949 



PCT/US99/04003 



Claims 

We claim: 

1. A method for modulating the effects on cells of a protein selected from the 
group consisting of growth and differentiation factor 8 [GDF-8] and bone 
morphogenetic protein 1 1 [BMP-1 1], said method comprising administering to said 
cells an effective amount of foUistatin. 

2. The method of claim 1, wherein the protein is GDF-8. 

3. The method of claim 1, wherein the protein is BMP- 11. 

4. A method for blocking the effects on cells of a protein selected from the 
group consisting of growth and differentiation factor 8 [GDF-8] and bone 
morphogenetic protein 1 1 [BMP-1 1], said method comprising administering to said 
cells an effective amount of follistatin. 

5. The method of claim 4, wherin the protein is GDF-8, 

6. The method of claim 4, wherin the protein is BMP-1 1. 

7. A method for treating a disorder associated with neural or muscular effects 
of a protein selected from the group consisting of growth and differentiation factor 8 
[GDF-8] and bone morphogenetic protein 11 [BMP-1 1], said method comprising 
administering to said cells an effective amount of follistatin. 

8. The method of claim 7, wherein the protein is GDF-8. 

9. The method of claim 7, wherein the protein is BMP^l 1 . 



10 



wo 99/45949 



PCT/US99/04003 



SEQUENCE LISTING 



(1) GENERAL INFORMATION: 

(i) APPLICANT: WOOD, Clive R. 

FITZ, LORI 

(ii) TITLE OF INVENTION: USE OF FOLLISTATIN TO MODULATE GROWTH 
AND DIFFERENTIATION FACTOR-8 [GDF-8] AND BONE 
MORPHOGENETIC PROTEIN [BMP-11] 

(iii) NUMBER OF SEQUENCES: 5 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: GENETICS INSTITUTE, INC. 

(B) STREET: 87 Cambridge Park Drive 

(C) CITY: Cambridge 

(D) STATE: Massachusetts 

(E) COUNTRY: USA 
<F) ZIP: 02140 

(V) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

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

(D) SOFTWARE: Patent In Release #1.0, Version #1.30 

(vi) CURRENT APPLICATION DATA: 

(A) APPLICATION NUMBER: 

(B) FILING DATE: herewith 

(C) CLASSIFICATION: 

(viii) ATTORNEY /AGENT INFORMATION: 

(A) NAME: LAZAR, STEVEN R. 

(B) REGISTRATION NUMBER: 32,618 

(C) REFERENCE /DOCKET NUMBER: GI 5327-PCT 

(ix) TELECOMMUNICATION INFORMATION: 

(A) . TELEPHONE: (617) 665-8260 

(B) TELEFAX: (617) 876-5851 



(2) INFORMATION FOR SEQ ID N0:1: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 20 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



1 



wo 99/45949 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: 
CCAGGATGGT CCGCGCGAGG 
(2) INFORMATION FOR SEQ ID NO : 2 : 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 20 base pairs 

(B) TYPE; nucleic acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY; linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0;2; 
TCAGTTGCAA GATCCGGAGT 
(2) INFORMATION FOR SEQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 23 base pairs 

(B) TYPE; nucleic acid 

(C) STRANDEDNESS; single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 
TCATTTGATA CACTTTCCCT CAT 
( 2 ) INFORMATION FOR SEQ ID NO ; 4 : 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 23 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(xi) SEQUENCE DESCRIPTION; SEQ ID NO: 4 
TCACTTTTTA CATCTGCCTT GGT 
(2) INFORMATION FOR SEQ ID NO: 5; 



wo 99/45949 



PCT/US99/04003 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 23 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : . single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: DNA (genomic) 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 : 
TCATTCTTTA CAGGGGATGC ACT 23 



3 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCX 

INTERNATIONAL APPLICATION PUBUSHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification ^ : 
A61K 38/17, C12N 5/00 



A3 



(11) InternaUonal Publication Number: WO 99/45949 

(43) International Publication Date: 16 September 1999 (16.09.99) 



(21) International Application Number: PCT/US99/04003 

(22) international Filing Date: 24 February 1999 (24.02.99) 



(30) Priority Data: 

09/037,118 



9 March 1998 (09.03.98) 



US 



(71) Applicant: GENETICS INSTITUTE, INC. [US/US]; 87 Cam- 

bridgePark Drive, Cambridge, MA 02140 (US). 

(72) Inventors: WOOD, Oive, R.; 2 Hawthorne Place #17R, 

Boston, MA 021 14 (US). FITZ, Lori, Jo; 13 Palmer Street, 
Arlington, MA 02174 (US). 

(74) Agent: LAZAR, Steven, R.; American Home Products Corpo- 
ration, Legal Affairs, Patent and Trademark Dept.-2B, One 
Campus Drive, Atm.: Kay E. Brady, Parsippany, NJ 07054 
(US). 



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



Published 

With international search report 
Before the expiration of the time limit for amending the claims 
and to be republished in the event of the receipt of amendments, 

(88) Date of publication of the international search report: 

18 November 1999(18.11.99) 



09 

m 

r- 
m 

o 
O 

■D 

<. 



(54) Title: USE OF FOLLISTATIN TO MODULATE GDF-8 AND BMP-1 1 
(57) Abstract 

Methods are provided for tlie modulation of the effects of GDF-8 and BMP-1 1, particularly on neural and muscular disoiders 
administration of follistatin for treating neural, muscle, disorders which are characterized by an abnormality in the levels or activity of 
GDF-8 or BMP-1 1. 



FOR THE PURPOSES OF INFORMATION ONLY 



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



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Arm»iia 


Fl 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


OA 


Gabon 


LV 


Latvia 


SZ 


Swaziland 


AZ 


Azeibaijan 


GB 


United Kmgdom 


MC 


Mcmaco 


TD 


Chad . 


BA 


Bosnia and Herzegovina 


G£ 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Tuitmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


UZ 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CG 


CCMlgO 


KE 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


ZW 


Zimbabwe 


CI 


Cdie d'l voire 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






ON 


China 


KR 


Republic of Korea 


PT 


Portugal 






CU 


Cuba 


KZ 


Kazakstan 


RO 


. Romania 






CZ 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


LI 


Liechtenstein 


SO 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


S£ 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







INTERNATIONAL SEARCH REPORT 



Internr" -<al ApplleMlon No 

PCT/uS 99/04003 



A. CLASSIFICATION OF SUBJECT MATTER . 

IPC 6 A61K38/17 C12N5/00 



According to International Paient Classttication (IPC) or to both nattonal classification and IPC 



B. FIELDS SEARCHED 



Minimum documentation searched (classificatton system followed by classification syrrd:>ot3) 

IPC 6 A61K 



Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched 



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



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category " Citatton of document, with indication, where appropriate, of the relevant passages 



Relevartt to claim No. 



THOMSEN G H: "Antagonism witriln and 
around the organizer: BMP inhibitors in 
vertebrate body patterning" 
TRENDS IN GENETICS, 

vol. 13, no. 6, 1 June 1997 (1997-06-01), 
page 209-211 XP004065308 

ISSN: 0168-9525 
the whole document 

WO 95 10611 A (HARVARD COLLEGE) 
20 April 1995 (1995-04-20) 
the whole document 



1-9 



US 5 700 911 A (CELESTE ANTHONY J 
23 December 1997 (1997-12-23) 
the whole document 



ET AL) 



1-9 



1-9 



-/- 



Furtfver documents are listed in the continuation of box C. 



Patent family members are listed in annex. 



Special categories of cited documents : 

"A" document deflnir^ the general state of the art which is not 

considered to be of particular relevance 
"E" earlier document but published on or after the international 

filing date 

T" document wtiich may throw doubts on priority claim(s) or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

*0" document referring to an oral disclosure, use, exhibition or 
other means 

"P" document published prior to the international fBing date but 
later than the priority date claimed 



T" later document published after the international filing date 
or priority date and not in conflict with the application but 
cited to understand the prirtcipte or theory underlying the 
invention 

"X" document of particular relevance; the claimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step when the document is taken atone 

"V" document of particular relevance; 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 art. 

"A" document member of the same patent family 



Date of the actual completion of the international search 



17 September 1999 



Date of mailing of the intematranal search report 



05/10/1999 



Name and mailing address of the ISA 

European Patent Office. P. B. 5818 Patentlaan 2 
NL -2280 HVRijswiik 
Tel. (+31-70) 340-2040. Tx. 31^51 epo nl. 
Fax: (+31-70) 340-3016 



Authorized officer 



Hagenmaier, S 



FofiTi PCT/ISA«10 (second 8he«t)(Jdy 1992) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



Internr" ^nal Appllcatton No 

PCT/US 99/04003 



C.(Continuatlon) DOCUMENTS CONSIDERED 70 BE RELEVANT 



Category ° Citation of document, with indication, wtiere appropriate, of ttie relevant passages 



Relevant to claim No. 



wo 94 26892 A (GENETICS INST) 
24 November 1994 (1994-11-24) 
the whole document 

WO 94 21681 A (UNIV JOHNS HOPKINS MED ;LEE 
SE JIN (US); MCPHERRON ALEXANDRA C (US) 
29 September 1994 (1994-09-29) 
the whole document 

A FAINSOD ET AL: "THE DORSALIZING AND 

NEURAL INDUCING GENE FOLLISTATIN IS AN 

ANTAGONIST OF BMP-4" 

MECHANISMS OF DEVELOPMENT, 

vol. 1, no. 63, 1 April 1997 (1997-04-01), 

page 39 50 XP002076023 

ISSN: 0925-4773 
the whole document 

68 2 306 481 A (UNIV MANCHESTER) 
7 May 1997 (1997-05-07) 
the whole document 

US 5 545 616 A (WOODRUFF TERESA K) 
13 August 1996 (1996-08-13) 
the whole document 

GAMER ET AL.: "A NOVEL BMP EXPRESSED IN 
DEVELOPING MOUSE LIMB, SPINAL CORD, AND 
TAIL BUD IS A POTENT MESODERM INDUCER IN 
XENOPUS EMBRYOS" 
DEVELOPMENTAL BIOLOGY, 
vol. 208, April 1999 (1999-04), pages 
222-232, XP002115687 
the whole document 



1-9 



1-9 



1-9 



1-9 



form PCT/I&A/210 (contimjatton of second sheet) (July 1982) 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 



\r\\ itional application No. 

PCT/US 99/04003 



Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) 

This Internationa) Search Report has not been established in respect ol certain claims under Article 17(2)(a) tor the following reasons: 



1. X Claims Nos.: 

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

Remark: Although claims 7-9 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. 

2. I I Claims Nos.: 

because they relate to parts of the International Application that do not comply with the prescribed requirements to such 
an extent that no meaningful International Search can be carried out, specifically: 



3. I I Claims Nos.: 

because they are dependent claims and are not dratted in accordance with the second and third sentences of Rule 6.4<a). 

Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) 

This International Searching Authority found multiple inventions in this international application, as follows: 



f I As all required additional search fees were timely paid by the applicant, this International Search Report covers all 
' — ' searchable claims. 



2- I I As alt searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment 
of any additional fee. 



3. I As 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 fvios.: 



\^ 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 [ | The additional search fees were accompanied by the applicant's protest. 

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



Form PCT/ISA/210 (continuation of first sheet (1)) (July 1998) 



INTERNATIONAL SEARCH REPORT 

jrmatlon on patent family member* 



Intern'' nal Application No 

PCT/US 99/04003 



Patent document 
cited in search report 


Publication 
date 


Patent family 
member<s) 


Putilication 
date 


WO 9510611 A 


20-04-1995 


AU 


701623 B 


04-02-1999 






AU 


7980694 A 


04-05-1995 






CA 


2174098 A 


20-04-1995 






EP 


0726948 A 


21-08-1996 






JP 


9503673 T 


15-04-1997 



US 5700911 A 23-12-1997 



US 


5639638 A 


17-06- 


1997 


AU 


678582 6 


05-06- 


1997 


AU 


6910594 A 


12-12- 


•1994 


BR 


9406715 A 


06-02- 


■1996 


EP 


0698094 A 


28-02- 


•1996 


FI 


955419 A 


08-01- 


•1996 


JP 


9501304 T 


10-02- 


1997 


NO 


954492 A 


08-11- 


1995 


UO 


9426892 A 


24-11- 


•1997 



WO 9426892 A 24-11-1994 



AU 


678582 B 


05-06-1997 


AU 


6910594 A 


12-12-1994 


BR 


9406715 A 


06-02-1996 


EP 


0698094 A 


28-02-1996 


FI 


955419 A 


08-01-1996 


JP 


9501304 T 


10-02-1997 


NO 


954492 A 


08-11-1995 


US 


5639638 A 


17-06-1997 


US 


5700911 A 


23-12-1997 



UO 


9421681 


A 


29-09-1994 


CA 


2157577 A 


29-09-1994 










EP 


0690873 A 


10-01-1996 










JP 


9507829 T 


12-08-1997 










US 


5827733 A 


27-10-1998 


GB 


2306481 


A 


07-05-1997 


AU 


7313896 A 


15-05-1997 










CA 


2235412 A 


01-05-1997 










EP 


0855916 A 


05-08-1998 










WO 


9715321 A 


01-05-1997 



US 5545616 A 13-08-1996 NONE 



Form PCT/tSA/210 (patent family annex) (July 1992) 



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