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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCX 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification ^ : 
A61K 38A7, C12N 5/00 



A2 



(11) International Publication Number: WO 99/4S949 

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



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

(22) International Filing I>ate: 24 February 1999 (24.02.99) 



(30) Priority Data: 

09/037,118 



9 March 1998 (09.03.98) 



US 



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

bridgcPark Drivc» Cambridge. MA 02140 (US). 

(72) Inventors: WOOD. Clivc, 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, 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, FT, 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, FI, FR, GB. GR, IE. IT, 
LU. MC, NL, FT. SE), OAPl 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-11. particularly 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-11. 



FOR THE PURPOSES OF INFORMATION ONLY 



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



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Annenia 


Fl 


Finland 


LT 


Lithuania 




Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azeifoaijan 


GB 


United Kingdnn 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


G«»gia 


, MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belsiuin 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


IXnkmenistan 


BF 


Burtina Paso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


EG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


XT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mmigolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BV 


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 


Vict Nam 


CG 


Congo 


KE 


l^ya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


ZW 


Zimbabvre 


CI 


COtc d'lvoire 


KP 


Demoaatic 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 






C2 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


U 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


BG 


Singf^pore 







wo 99/45949 



PCTAJS99/04003 



USE OF FOLLISTATIN TO MODULATE GDM AND BMP-1 1 

'5 

FTFT n OF THE INVENTION 
The present invention relates to use of foUistatin 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, Cell, 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. Cbem. 267:18924-18928 (1992). Ebner et al., Science , 260:1344-1348 
(1993) describe the existence of Type I and Type H TGF-p receptore, and the effects of 
25 the Type I receptor on binding of TGF-p to the Type II 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 teen 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 INVENTION 

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 amount of follistatin. 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 
follistatin. 

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

15 production and/or activity of ^DF-8 or BMP-1 1, thereby affecting the growth, 
formation, differentiation and maintenance of cells using a follistatin protein, or a DNA 
molecule encoding a follistatin 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 treatment of diseases 

20 and disorders involving neural or neuronal and muscle cells 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 follistatin for the treatment of traumatic or chronic injury to the spinal cord, 

25 or to the nerve or muscle system. 

DFT An D ESCRIPTION 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 ceUs. 

Two forms of follistatin (FS) are produced as a result of alternative splicing. 
These forms are FS-288 and FS-315. The FS-SIS 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 
S 268:15579(1993)). Recombinant forms of each of these molecules are expected to have 
different properties (Sumitomo et al., Biochem. Biophvs. Acta 208: If 1995)) and are 
envisaged to be useful for inhibiting the action of GDF-8 and BMP-1 1 . 

The expected properties of foUistatin, in light of the present showing, include 
differentia] 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 may be suboptimal m 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)). 

FoUistatin 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 foUistatin 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 Exanq)le 2 below. The foUistatin 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), vasctilar disorders,, trauma, metaboUc d^mgements, 
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-Hof&nann disease), epilepsy, syringomyelia, peripheral neurof^y, congenital 
anomalies and tumors. Muscle-related conditions for treatment include without 
limitation rhuscular 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 PCTAJS99/04003 
5 myotonia congenital, familial periodic paralysis, paroxysmal myoglobinuria, myasthenia 
gravis, Eaton-Lambert syndrome, secondary myasthenia, denervation atrophy. 

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

10 disclosed in Robertson et al., Biochem. Biophvs, Res. Commun. 149:744-749 (1987). 
The disclosures of each of these publications is hereby incorporated by reference herein. 
In addition, truncated polypeptides which comprise partial fragments of the full 
follistatin 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 follistatin 

13 sequences, which maintain the ability 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 follistatin polypeptide as a functional fragment of follistatin 
may readily be determined, for example, using the assay described in Example 2. 

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

20 This includes the fusion of FS-288, FS-3 15 or FS-303 sequences with the hinge, CH2 
and CH3 domains of a himian inununoglobulin gamma isotype, e.g., ganmia 1 or 4. 
Such a fusion protein is expected to produce a dimeric molecule, with the improved 
pharmacokinetics expected for an inmiunoglobulin Fc fusion. In addition, the constant 
domains or secretory tailpieces of alpha or mu immunoglobulin 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 ther^^utic utilities. The human FS gene contains four domains 
each encoded on a separate exon, in addition to an exon encoding a N-tenninal 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 follistatin proteins 

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



4 



wo 99/45949 PCT/US99/04003 

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 

iO 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 (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 
foUistatin polypeptides encoded thereby are also usefiil for the present invention. 

The present invention may include gene therapy, in which transfeciion of ceDs 

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

25 8 or BMP-1 1 in an organism or ceU. The increased foUistatin 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. 

FoUistatin 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 foUistatin may be incorporated into a column 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 foUistatin or functional fragments thereof are used to bind to the GDF-8 
or BMP-1 1, and later subjected to conditions which result 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., interieukins and CSFs) and antibiotics, may also optionally be 
included in or administered simultaneously or sequentially with, : the Fbllisiatin 
composition in the methods of the invention. 

There is a wide range of methods which can be used to deliver the cells 
expressing follistatin proteins to a site for use in modulating a GDF-8 or BMP- 11 
response. In one embodiment of the invention, the cells expressing follistatin 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 prefened embodiment, the cells expressing follistatin 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 
cells into the solid matrix, and hold them there while allowing proliferation of the cells. 

In a method of the present invention, the cells expressing follistatin 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 follistatin. The BMP may be applied in manners known in the art, such as 
described in the above patents, as well as in United States Patent 5,171,'579, the 
disclosure of which is also hereby incorporated by reference. 
Expression of Follistatin Protein 

In order to produce follistatin protein, the DNA encoding the desired protein is 
transferred into an appropriate expression vector and introduced into mammalian cells 
or other preferred eukaiyotic or prokaryotic hosts by conventional genetic engineering 



wo 99/45949 PCT/US99/04003 

5 techniques. The presently preferred expression system for biologically active 
recombinant follisiatin 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 toahose skilled in the art upon consideration of these descriptions. Those 

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



EXAMPLES 

EXAMPLE 1. BLicore 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 
(RU) of follistatin were immobilized by this procedure. Purified BMP and GDF 

20 proteins were each inj«:ted over the immobilized follistatin for 10 minutes at 2 jil/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 temperature 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 bindmg 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 

S 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 

10 transferred to fresh media without protein. The animal caps were cultured overnight 
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 mesoderm 
15 (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). FoUistatin 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 
20 follistatin (BMP-1 1 50ng/ml and 500ng/ml follistatin). Together, the Biacore binding 
results and inhibition on the Xenopus animal cap assay demonstr^e that follistatin is 
an antagonist 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 Follistalin 

25 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 1 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 identiiy the 

30 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- 

35 primed first strand cDNA synthesis fvom primary tissue poly A+ RNA (e.g., from ovary 



wo 99/45949 PCt/US99/04003 

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-fonvard. The reverse primer for generating 1-5 

1 5 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'-dCCAGGATGGTCCGCGCGAGG-3' [SEQIDNO:!] 
FS-reverse 5: 5'-dTCAGTTGCAAGATCCGGAGT-3' [SEQ ID NO:2] 
FS-reverse 4: 5'-dTCAnTGATACACTTTCCCTCAT-3' (SEQ ID NO:3] 
FS-reverse 3: 5'-dTCACTrTTTACATCTGCCrrGGT-3' [SEQ ID NO:4] 

25 FS-reverse 2: 5'-dTCATTCnTACAGGGGATGCAGT-3' [SEQ ID NO: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 fragments 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 construct 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 tGDF-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- 1 1 , 

4. A method for blocking the effects on cells of a protein selected from the 
group consisting of growth and differentiation factor 8 tGDF-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-81 and bone morphogenetic protein 11 [BMP-11], 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-1 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 CambridgePark 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 #1.3 0 

(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) €65-8260 

(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 NO: 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 AGT 23 



3 



WORLD FNTCLLECTUAL PROPERTY ORGANIZATION 
Intemational Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



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


A3 


(11) International Publication Number: WO 99/45949 
(43) International Publication Date: 16 September 1999 (16.09.99) 


(21) Intemational Application Number: PCr/US99/04003 

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

(30) Priority Data: 

09/037.1 18 9 March 1998 (09.03.98) US 

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

bridgcParic Drive. Cambridge. MA 02140 (US). 

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

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

(^^\ AcTi>iit* T A 7 AH ^fpv^n R ' Amf*'rif*{iTi Hnmp ^fv\Hii/*tc f^r\mf\- 
\t*fj /\KCUl> l^/\£<nJ\f OlCVCIl. IV.. nllldlCoIl JiUIIlC x lUUUClo V^UJ uO~ 

ration, Legal Affairs. Patent and Trademark Dept.-2B, One 
Campus Drive. Attn.: Kay E. Brady. Parsippany, NJ (J7054 
(US). 


(81) Designated States: AL. AM. AT. AU. A2. 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. IT. 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, R. 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 intemational 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) 



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



(57) Abstract 

Methods are provided for the modulation of the efTects of GDF-8 and BMP-1 1. particularly on neural and muscular disoideis 
administration of follistatin for treating neural, muscle, disorders which are characterized by an abnonnality 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 


^)ain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Annenia 


FI 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FK 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azeibaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Heizcgovina 


G£ 


Georgia 


MD 


Republic of Moklova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


1\iikey 


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 


Vict Nam 


CC 


Congo 


K£ 


Kenya 


NL 


NetherlaiKls 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyiigyzstan 


NO 


Norway 


ZW 


Zimbabwe 


CI 


CGte d'l voire 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Poroigal 






CU 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






CZ 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Gennany 


LI 


Liechtenstein 


SD 


Sudan 






DK 


Denmaric 


LK 


Sri Lanka 


S£ 


Sweden 






EE 


Estonia 


LR 


Liberia 


1SG 


Sing2^rc 







INTERNATIONAL SEARCH REPORT 



(nternr^* lal Application No 

PCT/uS 99/04003 



A. CLASSIFICATION OF SUBJECT MATTER , 

IPC 6 A61K38/17 C12N5/00 



According to International Patent ClassHication (IPC) or to both nattonal dassittcation and iPC 



B. FIELDS SEARCHED 



Minimum documentation searched (ciaeaiticatton system followed by classification syrr^ls) 

IPC 6 A61K 



Ooeumentation searched other than minimum Oocumentation to the extent that such documents are included in the lields searched 



Electronic data Dase consulted during the International search (name ol data t>ase and. wtiere practical, search terms used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category * Citation of documertt. with indication, whais appropriate, of the relevant passages 



Relevant to daim No. 



THOMSEN G H: "Antagonism within 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 

UO 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 



-/- 



m 



Further documents are listed in the continuation of box C. 



Patent family members are listed in annex. 



* Special categories of cited documents : 

'A" document defining the general state of the an which is not 

considered to be of particular relevance 
"E* eartter document but published on or after the International 

filing date 

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

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

T" document published prior to the international faing date but 
later than the priority date claimed 



T" later document published after the international filir>Q date 
or pnority date and not in conflict with the application but 
cited to understand the principle or theory undertying the 
invention 

"X- document of particular relevance: the ciaimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step when the document is taken alone 

"Y" 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 siciPed 
in the art. 

document member of the same patent family 



Date of the actual completion of the intemationat search 

17 September 1999 


Date of mailing of the international search report 

05/10/1999 


Name and mailing address of the ISA 

European Patent Office. P.B. 581 8 Patenttaan 2 
NL-2260 HV Rijswijk 
Tel. (+31-70) 340-2040, Tx. 31 651 epo nl. 
Fax: (+31-70) 340-3016 


Authorized officer 

Hagenmaier, S 



Form PCT/tSACiO (second sheet) (July 1 992) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



Intcrnr" ^nal Appllc«tion No 

PCT/US 99/04003 



C.(Contlnuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT 



Category ° Citation ot document, witfi indication. where appropriate, of the relevant pas&ages 



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 

GB 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, XP002 115687 
the whole document 



1-9 



1-9 



1-9 



1-9 



Form PCT/ISA/210 (continuation of socond fiheet) Uiiy 1992) 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 



tni ttional application No. 

PCT/US 99/04003 



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

This intemationai Search Report has not been established in respect ol certain claims under Article 17(2)(a) for 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. 



□ 



Claims Nos.: 

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



3. I I Claims Nos.: 

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

Box 11 Observations where unity of Invention is lacking (Continuation of Item 2 of first sheet) 

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



1 . I 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 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 tees were paid, specificalty claims Nos.: 



^' CZI fequired 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 accofT>panied by the applicant's protest. 

[ I No protest accompanied the payment of additional search tees. 



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



INTERNATIONAL SEARCH REPORT 

jnnstlon on patent famtty members 



Intemr naj Application No 

PCT/US 99/04003 



Patent ckxsument 


Publication 




Patent family 


Publication 


cited in search report 


date 




memt)er(s) 


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 

X i vU X 77 / 






AU 


678582 B 


05-06-1997 

w9 UU X 77 / 






AU 


6910594 A 


X£. XC J.77H 






BR 


9406715 A 


06-02-1996 






EP 


0698094 A 


28-02-1996 






FT 

r X 


95S41Q A 


I/O VI X 77V 






JP 


9501304 T 


lU Ub X77/ 






NO 


954492 A 


I/O 11 1 773 






WW 




1-1 QQ7 

11 1 77/ 


WO 9426892 A 


24-11-1994 


AU 


678582 8 


1/3 UU 177/ 






AU 


6910^94 A 


It 1£. 177*T 






BR 


9406715 A 


vU V/C 1770 






EP 


0698094 A 


28-02-1996 






FI 


955419 A 


08-01-1996 








7«>U10vH 1 








NO 


QR44QP A 


wO 11 1 773 










1/ V/U 177/ 






us 


5700911 A 


23-12-1997 


WO 9421681 A 


2Q-0Q-10Q4 


TA 




t7 U7 177H 






FP 




10-01 -IQQfi 

Iw 1/ 1 1770 






ilP 


0^07ftP9 T 

79 w / 0£.7 1 


l£. VO 177/ 










l/ lU 1770 


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«lO (patent family annex) <July 1992) 



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