CA2222609A1 - Methods and uses of connective tissue growth factor as an induction agent - Google Patents

Abstract

The present invention relates to novel methods and compositions related to the administration of connective tissue growth factor, alone or in combination with other growth factors, compositions or compounds, to induce the formation of connective tissue, including bone, cartilage, and the skin.

Description

~o~ AND ~8ES OF
N ~ lV~ TI8SUE GROWTH FACTOR
A8 AN lN~ lON AGENT

The information disclosed in this Specification was made in part with Government support by grant no. GM 37223, awarded by the National Institute of Health. The government may have certain rights in the invention disclosed in this Specification.

l. 8TA ~l~ OF RELATED CA8E8 This application is related to and is a continuation-in-part application of Serial Number 08/459,717, entitled "Connective Tissue Growth Factor," filed June 2, 1995, which is a continuation-in-part application of Serial No.
08/386,680, filed on February 10, 1995, having the same title, which is a divisional application of Serial Number 08/167,628, filed December 14, 1993, now issued as U.S.
Patent No. 5,408,040, which is a continuation of Serial No.
07/752,427, filed on August 30, 1991, now AhAn~oned.

2. FIELD OF THE l~V ~:~. ' ON
This invention relates generally to the field of growth factors and specifically to Conn~ctive Tissue Growth Factor (CTGF) and methods of use thereof.

3 R~Q D OF TH~ lNV~. lON
A. The Role Of Growth F~ctors In Bone And Cartilage Formation Bone And Cartilage Formation. The formation of tissue and organs in all multicellular orgAn;! - that arise from a single fertilized egg requires the differentiation of specialized cell types from non-differentiated stem cells.
As embryogenesis proceeds, more highly specialized cell types and complex structures are formed. Currently, however, little concrete information is available on the identification of W O96/38168 PCTrUS96/08210 the specific factors or the ?~h~n;! of action of these factors on skeletal or cartilage formation in vertebrate ~n; -1~, including humans.
There are two common types of bone formation in the - -l;an system: intr~ h~anous ossification and endo~hon~al ossification. The formation of the bones of the skull are an example of intramembranous ossification. There, mesenchymal cells from the neural crest interact with the extracellular matrix of the cranial epithelial cells and form bone. Hall, Amer. sci., 1988, 76174-181. Mesenchymal cells condense into small islands and differentiate into osteoblasts and capillaries. The osteoblasts secrete a specific type of extracellular matrix, (osteoid) which is capable of binding calcium salts.
Endochondral ossification is the process by which the long bones of the axial skeleton (arms and legs), and the vertebra and ribs form. Hall, supra. During this process the formation of bone occurs via a cartilaginous tissue inteL ~ te stage. In ~ ~ls, the long bones form from certain mesenchymal cells in the embryonic limb buds. These cells form chondrocytes, and secrete a cartilaginous matrix.
Other s~lL~u.lding s~nchymal cells form the perichondrium (ultimately, the periosteum). In some cases, chondrocytes adjacent to the region where chondrocytes are proliferating and forming differentiate into hypertrophic chondrocytes.
Hypertrophic chondrocytes produce a different type of matrix, and alter their tissue orientation to form the physis. The structure of the physis is arranged in multiple cellular columns c~ ~sed of zones of cellular hypertrophy, proliferation, ossification and vascularization. Hall, supra i Gilbert, "Transcriptional regulation of gene expression," DEVELOPMENTAL BIOLOGY, 5th ed. Sinaur Assoc., p.
387-390 (1994). This results in a gradation of cell transformation from chondrocytes to osteoblasts which form the mineralized bone.
Endochondral ossification is an active, ongoing process that occurs in ~ ~ls during the growth from infant to adult. The differentiation of mesenchymal cells to W O96/38168 PCTrUS96/08210 chondrocytes, their proliferation and replacement by osteoblasts are dependent on growth factors (including the TGF-~ family), and on the mineralization of the matrix.
Tuan, 1984, J. Exp. Zool. (suppl.) 1:1-13 (1984); Syfestad and Caplan, 1984, Devel. Biol . 104:348-386.
With regard to connective tissue, it is felt that all skeletal elements in mammals are derived from a single stem cell that is capable of differentiating into the specific cell types that compose muscle, cartilage, bone and tendon.
These cells also appear to be capable of differentiating into adipose tissue.
The Relevant Art Related To Growth Factors And The Formation Of Bone And Cart~1age. Prior to the present invention, it was known generally that growth factors comprise a class of secreted polypeptides that st; l~te target cells to proliferate, differentiate and organize developing tissues. Typically, a growth factor's activity is dependent on its ability to bind to specific receptors, thereby st; l~ting a signaling event within the cell.
Examples of some well-studied growth factors include platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transfo~ ;ng growth factor beta family (TGF-~), transforming growth factor alpha (TGF-~), epidermal growth factor (EGF), and fibroblast growth factors (FGF).
Effect Of TGF-~ On rho~ocyte Growth, Differentiation and Cartilage Formation. The TGF-~s play a role in chon~ogenesis. As previously reported, TGF-~l and TGF-~2 increase chondrogenesis in embryonic rat mesenchymal cells (Seyedin,et al.., 1987, J. Biol. Chem. 262: 1946-1947), and either isoform can induce formation of chondroblasts from murine muscle mesenchymal cells in culture. Seyedin. et al., 1986, J. Biol. Chem. 261: 5693-5695. Application of the TGF-~s to murine embryonic prechondroid tissues increases differentiation of mesenchymal cells, production of proteoglycans, and replication of chondroblasts. Centrella, et al., 1994, Endocrine Reviews 15:27-38; Thorp and Jakowlew, 1994, Bone 15: 59-64.

W O96/38168 PCTrUS96/08210 Using in-situ hybridization, decreased levels of TGF-~3 were found in the growth plates of ~n; ~1 S with three separate disorders where chondrocytes cease to differentiate.
Id. In organ cultures of bovine articular cartilage, type II
collagen and proteoglycan synthesis were increased after TGF-at~ ; n; ~tration. Morales and Roberts, 1988, J. Biol . Chem.
263: 12828- 12831. In contrast, the TGF-~s have been shown to decrease expression of type II and type X cartilage-specific collagens, synthesis of chondrocyte proteoglycans, and activity of alkaline phosphatase in cultured chondroid cells.
Mundy. The effects of TGF-~ on bone," Clinical Applications of TGF-~., 1991, Wiley Chichester, Ciba Foundation Symposium 157: 137-151. Rabbit growth plate chondrocyte differentiation is inhibited by TGF-~, while growth plate chondrocyte mitogenesis is increased. Kato, et al., 1988, Proc. Natl. Acad. Sci. USA 85: 9552-9556. In addition, large concentrations of TGF-~l or TGF-~2 added to an osteoinductive model favor cartilage, rather than the preference for ~one formation, when smaller doses are used. Mundy, supra. This aç~ tion of apparently contradictory data has hindered efforts to define a function for the TGF-~s in chondrogenesis.
The Bone Mo~phogenic Proteins And Bo~e Formation. A
family of proteins termed the bone morphogenetic proteins (BMP's are capable of i~ducing ectopic bone formation in certain ~ -lian species. With the exception of BMP-l, which encodes a metalloprotease, all of these proteins have structures that are related to TGF-~. However, it is not known which, if any of the BMP's are responsible for the regulation of bone formation during normal embryogenesis.
BMP's were first isolated from ~f~;n~ralized bone as factors that induced bone at extra skeletal ectopic sites.
Three peptides were originally identified as BMP-1, BMP-2A, and BMP-3. Celeste, et al., 1990, Proc. Natl Acad. Sci. USA
87: 9843-9847; Kubler and Urist, 1990, Clin. orthopedics and Rel. Res. 258: 279-294. The latter two BMPs are members of the TGF--,~ superfamily. Subsequently, five more closely related ~-h~rs of the BMP group have been identified and _ -W O96/38168 PCTrUS96/08210 cloned. BMP-5, BMP-6, and BMP-7 are most similar to vgr/60A, while BMP-2 and BMP-4 are more similar to Decapentaplegic.
Both vga/60A and Decapentaplegic are Drosophila genes that control dorsal/ventral axis pattern formation. Hoffman, 1992, Mol. Repro and Dev. 32: 173-178.
In-situ hybridization has localized BMP's gene transcription to areas of bone formation in the limb bud at specific times during development, suggesting a physiologic role. The BMPs ;n~l~ce adventitial post-fetal mesenchymal cells to switch from fibrogenetic to ~hon~roosteoprogenetic patterning. Kubler and Urist, supra. Several lines of data suggest the BMPs may act synergistically with TGF-~s to initiate the cascade of osteoinduction in-vivo. In murine subcutis, TGF-~l ~nh~nce$ the production of ectopic bone by most BMPs. BMP-6 (also known as VGR-l) is expressed in hypertrophic cartilage at the same time and in the same areas as the TGF-~s, and is associated with collagen type X
expression. See, Celeste, et al., supra.
The addition of TGF~-2 to bone explants which have been treated with either BMP-2 or BMP-3 results in increased osteoinductive activity and an increased ratio of cartilage to bone when ~l _~ed to either factor alone. Bentz, et al., 1991, Matrix 11:269-275. However, the synergistic effect of these proteins by the TGF-~s is not universal. TGF-~l has been shown to directly decrease BMP-2 expression in fetal rat calvaria cultures. Harris, et al., 1994, ~. Bone and Mineral Res. 9: 855-863. Since BMP-2 is apparently important in bone cell differentiation, it has been suggested that TGF-~l may be acting as a switch to monitor the differentiation fates of chondro- or osteo- blastic precursors.
Other Factors Found To Be Expressed Tn Developing Tissue. Cyr61 is a growth regulator which has been found to be expressed in developing mouse embryo and extraembryonic - tissues. O'Brien and Lau, 1992, Cell Growth Differ. 3:645-654. Cyr61 is related to but distinct from CTGF and prior to ~ the instant invention, the specific activity of Cyr61 was not known.

CA 02222609 1997~ 27 W os6/38168 pcTrus96lo82lo B. The Rolo Of Growth Factors In Wound ~9~1 i ng Platelet Derived Growth Factor And Wo7lnd ~e~7 ;ng, PDGF is a dimeric molecule consisting of an A chain and a B
chain. The Ch~; n~ form heterodimers of homodimers and all combinations isolated to date are biologically active. With respect to the factor's activity, PDGF has been characterized as a cationic, heat-stable protein found in the ~-granules of circulating platelets. The molecule has been further characterized as a mitogen and a chemotactic agent for connective tissue cells such as fibroblasts and smooth muscle cells.
Because of PDGF's biological activity and release during wound healing, PDGF has been identified as a growth factor involved in wound healing, as well as pathological conditions showing on overproduction of connective tissue, including atherosclerosis and fibrotic diseases.
It has been hypothesized that growth factors other than PDGF may play a role in the normal development, growth, and repair of human tissue.
TGF-~ And Wound ~e~7 ing, The formation of new and regenerating tissue requires the coordinate regulation of various genes that produce both regulatory and structural molecules which participate in the process of cell growth and tissue organization. As with bone induction, it appears that TGF-~ plays a central regulatory component of this process.
TGF-~ is released by platelets, macrophages and neutrophils which are present in the initial phases of the repair process. TGF-~ can act as a growth st; -l~tory factor for mesenchymal cells and as a growth inhibitory factor for endothelial and epithelial cells. It has been suggested that the growth stimulatory action of TGF-~ appears to be mediated via an indirect ?~-h~ni~ involving the induction of other genes including growth factors such as PDGF.
Several h~rS of the TGF-~ superfamily possess activities suggesting possible applications for the treatment of cell proliferative disorders, such as c~nc~. In particular, TGF-~ has been shown to be potent growth inhibitor for a variety of cell types (Massague, 1987, Cell W O96/38168 PCTrUS96/Q8210 49:437), MIS has been shown to inhibit the growth of human endometrial carcinoma tumors in nude mice (Donahoe, et al., 1981, Ann. Surg. 194:472), and inhibition has been shown to suppress the development of tumors both in the ovary and in the testis (Matzuk, et al., 1992, Nature, 360:313).
Many of the ~ h~s of the TGF-~ family are also important mediators of tissue repair. TGF-~ has been shown to have marked effects on the formation of collagen and causes of striking angiogenic response in the newborn mouse (Roberts, et al., 1986, Proc. Natl. Acad. Sci., USA 83:4167).
The bone morphogenic proteins (BMPs) can induce new bone growth and are effective for the treatment of fractures and other skeletal defects (Glowacki, et al., 1981 Lancet, 1:959;
Ferguson, et al., 1988, Clin. Orthoped. Relat. Res., 227:265;
Johnson, et al., 1988, Clin. Orthoped. Relat. Res., 230:257).

C. ronnective ~issue Growth Factor A previously unknown growth factor, related to PDGF, and termed Co~nective Tissue Growth Factor (CTGF), has been reported in a related patent. See, U.S. Patent No.

5,408,040. CTGF is a cysteine-rich mitogenic peptide which is selectively ;n~nc~ in fibroblasts after activation with TGF-~. Igarashi, et al., 1993, Mol. Biol. Cell 4: 637-645.
CTGF is a -- h~t- of a family of peptides that include serum induced gene products ceflO (Simmons, et al., 1989, Proc. Natl. Acad. Sci. USA 86:1178-1182), cyr61 (O'Brien, et al., 1990, Mol. Cell. Biol. 10:3569-3577), fispl2/IG M1 (Ryseck, et al., 1993, Cell Growth & Differ. 2:225-233), and a chicken transforming gene, nov (Joliot, et al. 1992, Mol.
Cell Biol. 12:10-21 (1992). CTGF also shares sequence homology with a drosophila gene product, twisted gastrulation (twg) (Mason, et al.., 1994, Genes & Develop. 8:1489-1501), which determines cell fates during dorsal/ventral pattern - formation in the embryo.
As reported in that patent, CTGF is the product of a distinct gene. As also reported in U.S. Patent No.
5,408,040, CTGF possesses mitogenic activity. The ultimate result of this mitogenic activity in vivo, is the growth of W O96/38168 PCTrUS~Gi'-~10 targeted tissue. CTGF also possesses chemotactic activity, which is the chemically induced v~ e~t of cells as a result of interaction with particular molecules.
Although the molecule is antigenically related to PDGF, there is little if any peptide sequence homology between CTGF
and PDGF. Anti-PDGF antibody has high affinity to the non-reduced forms of the PDGF isomers and the CTGF molecule and ten-fold less affinity to the re~llc~ forms of these peptides, which lack biological activity.
A second protein, identified as "connective tissue growth factor-2" or "CTGF-2," has been reported also. See, PCT Application No. PCT/US94/07736 (International Publication No. WO 96/01896). According to the PCT Application, CTGF-2 may also be used to ~nhAnc~ the repair of connective and support tissue. Although identified as a connective tissue growth factor, CTGF-2 is not closely related to the CTGF of the present invention. Specifically, the CTGF family is comprised of three distinct ylOU~ of proteins: CTGF/Fispl2, cyr61 and nov. The protein of the cl A; ~ invention falls within the first group of proteins, as compared to CTGF-2, which falls with the cyr61 group. PCT Application No.
PCT/US94/07736 at 4.
NotwithstAn~;ng the identification of various PDGF
related growth factors, including CTGF, prior to the present invention, such factors have not been proven to be an effective induction agent for the production of matrices, including the induction of bone and/or cartilage tissue.

4. 8UNMARY OF THE l~.V~ lON
The subject invention provides novel methods and compositions for the treatment of diseases, disorders or ailments wherein matrix and/or connective tissue production, including the production of bone and/or cartilage, is desired. The subject invention is likewise directed to the treatment of diseases, disorders or ailments wherein the promotion of wound healing is desired.
More specifically, the compositions of the present invention comprise CTGF and/or fragments and/or derivatives W O96138168 PCTrUS9''~210 thereof (hereinafter collectively "CTGF"), alone or in combination with other growth factors. The CTGF used in the subject compositions may be either obtained by isolation from natural sources, synthetic manufacture, or production by recombinant genetic engineering t~chn;ques.
In one aspect of the invention, the methods of the present invention comprise the administration of an effective amount of CTGF, alone or in combination with one or more compounds, to treat diseases, disorders or ailments wherein the induction of bone or cartilage tissue is desired. In a preferred embodiment of this method, such additional compound is a growth factor.
In another aspect of the invention, the methods of the present invention comprise the AC in;~tration of an effective amount of CTGF, alone or in combination with one or more compounds, again preferably one or more growth factors, to treat diseases, disorders or A;l ~nts wherein the ~l~ -Lion of wound heAl;ng is desired.
In a preferred embodiment of the invention, the composition comprising CTGF is A~' ; n;~tered directly onto or into the site in which bone or cartilage induction is desired so as to induce the formation of such bone or cartilage. In another embodiment, the composition is formulated for targeted delivery or alternatively, are designed for the release of the novel compositions in the relevant site (e.g., the wound in which cartilage formation is desired). In each case, the CTGF contAi n; ng composition is a~L~Liately fol ~lAted for Al ;n;~tration to a patient in need.

5. D~1N1~10NS
As used in this Specification, the term "CTGF" shall mean: (1) a protein encoded by the amino acid sequence set forth at Figure lC, (2) a protein having CTGF activity wherein such protein is encoded by the amino acid sequence of Figure lC wherein one or more amino acids have been added, deleted, mutated, substituted or otherwise altered ("derivative") and the nucleotide sequence encoding said protein can hybridize to the nucleic acid sequence of Figure _ 9 _ W O96/38168 PCTrUS96/08210 lC under stringent conditions, or (3) a fragment of CTGF or a derivative thereof.
As used in this Specification, the term ";n~-lr~," as used herein, shall mean to produce, form, to cause to produce, or to cause to form.
As used in this Specification, the phrase "induction agent" shall mean an agent, including proteins or other biological materials, which causes the production or formation of a specific end result (e.g., the production of connective tissue).
As used in this Specification, the term "polynucleotide"
denotes DNA, cDNA and/or RNA which encode untranslated sequences which flank the structural gene encoding CTGF. For example, a polynucleotide of the invention includes 5' regulatory nucleotide sequences and 3' untranslated sequences associated with the CTGF structural gene. A polynucleotide of the invention which includes the 5' and 3' untranslated region is illustrated in FIGURE lC. The S' regulatory region, including the promoter, is illustrated in FIGURE lB.
A more detailed description of the polynucleotides contemplated by the present invention may be found at U.S.
Patent No. 5,408,040.
As used in this Specification, the phrase "stringent conditions," as used herein, refers to those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M
sodium citrate/0.1% SDS at 50~C.; (2) employ during hybridization a denaturing agent such as fo1 ~ ;de, for example, 50% (vol/vol) foL - ;de with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42~C; or (3) employ 50% foL - ide, 5 x SSC (0.75 M
NaCl, 0.075 M Sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1~ SDS, and 10%
dextran sulfate at 42~C, with washes at 42~C in 0.2 x SSC and 0.1% SDS.
As used in this Specification, the phrase "recombinant expression vector" refers to a plasmid, virus or other W O96/38168 PCTrUS96/08210 vehicle known in the art that has been manipulated by insertion or incorporation of the CTGF genetic sequences.
As used in this Specification, the phrase "therapeutically effective" means that amount of CTGF which is effective in inducing bone or cartilage formation or wound healing.

6. BRIEF n~RTPTION OF THE DRaWINGS
FIGURE lA shows the structural organization of the CTGF
gene. Exons are indicated by boxed regions, with solid areas in the gene corresponding to the open reading frame.
FIGURE lB shows a c~ ~ison of nucleotide sequences between CTGF promoter and fisp-12 promoter. Identical nucleotides are marked with asterisks. The TATA box and other consensus sequences are indicated and ~h~ rl. The site of transcriptional initiation is indicated at position number +l .
FIGURE lC shows the complete nucleotide and deduced amino acid sequence for the CTGF structural gene and 5' and 3' untranslated sequences.
FIGURE 2 shows experimental in situ hybridization results related to the expression of CTGF transcripts in the growth plate of long bones in newborn mice. The in situ hybridization experiments were performed using an anti-sense CTGF RNA probe as described below. Chondrocytes in the proliferation zone are ~Llo~lyly positive for CTGF gene expression, indicating that CTGF is produced at sites of cartilage growth.
FIGURE 3 shows the expression of the CTGF gene during embryogenesis wherein a transgenic mouse is constructed using a fusion gene constructed from a CTGF promoter and a ~-galactosidase structural gene. This gene, introduced into the germ line, expresses ~-galactosidase at sites of CTGF
expression and can be detected by histochemical means by expressing sections of the developing transgenic ~n; -1 to the substrate X-gal which deposits a blue color at sites of ~-galactosidase activity. Panel A is a 12 day mouse embryo from such a transgenic mouse. The blue st~;n;ng is an area W O96/38168 PCTrUS~Gi'-~10 destined to become Meckel's cartilage, which is the first cartilage to form. Panel B is a photograph of the hind limb and paw which demonstrates st~;n;ng at the ends of the long bones and in the paw in the growth plates of the metatarsal.
FIGURE 4 provides evidence of induction of cartilage and bone in cultures of C3HlOT1/2 mouse embryonic stem cells.
C3HlOT1/2 cells were cultured as described under methods.
Cells were treated with either nothing (Panel A), 5 -azacytodine (Panel B), CTGF at 50 ng/ml (Panel C) or 5-azacyto~;n~ followed by CTGF (Panel D).
FIGURE 5 sets forth Northern blot analysis of CTGF gene expression in wound chambers implanted at sites of bone regeneration.
FIGURE 6 sets forth evidence related to the expression of CTGF in human osteoblasts in response to TGF-~.
FIGURES 7A-7D set forth results of a chon~rogenic assay.
FIGURE 7A provides the chondrogenic assay results for the control culture.
FIGURE 7B provides the ~hon~ogenic assay results for a culture in which 5 ng/ml TGF-~1 was added.
FIGURE 7C provides the chondrogenic assay results for a culture in which 5 ng/ml TGF-~1 and 10 ng cholera toxin were added.
FIGURE 7D provides the chondrogenic assay results for a culture in which 5 ng/ml TGF-~1, 10 ng/ml cholera toxin, and 5 ng/ml CTGF were added.
FIGURE 8A is a Scatchard Plot reflecting CTGF binding to NRK cells.
FIGURE 8B is a Sctachard Plot reflecting CTGF binding to rat ~hon~oblasts.

7, nT~TTT~n DESCRIPTION OF TUE lNV~L. lON
7.1. M~thods For M~; ng CTGF
Nucleic Acid Seq~encef~ ~nco~;ng CTGF.. In accordance with the invention, nucleotide sequences encoding CTGF or functional equivalents thereof may be used to generate recombinant DNA molecules that direct the expression of the protein or a functional equivalent thereof, in CA 02222609 l997-ll-27 W O96/38168 PCTrUS96/08210 a~o~iate host cells. Alternatively, nucleotide sequences which hybridize, under stringent position, to portions of the CTGF sequence may also be used in nucleic acid hybridization assays, Southern and Northern blot analyses, etc. In yet another method, DNA molecules encoding CTGF may be isolated by hybridization procedures comprising antibody screening of expression libraries to detect shared structural features.
Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be isolated and used in the practice of the invention for the cloning and expression of CTGF. Such DNA sequences include those which are capable of hybridizing to the human CTGF sequence under stringent conditions.
Altered DNA sequences which may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product. The gene product itself may contain deletions, additions or substitutions of amino acid residues within the CTGF sequence, which result in a silent change thus producing a functionally equivalent protein. Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hyd,~hilicity, and/or the amphipatic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having s; ;1~ hydrophilicity values include the following: leucine, isoleucine, valine; glycine, analine;
asparagine, glutamine; serine, threonine; phenyl~l~n;ne, tyrosine.
The DNA sequences of the invention may be engineered in order to alter the protein's sequence for a variety of ends including but not limited to alterations which modify processing and expression of the gene product. For example, mutations may be introduced using t~c-hn;ques which are well known in the art, e.g., site-directed mutagenesis to, for W O96/38168 PCTrUS96/08210 example, insert new restriction sites. For example, in certain expression systems such as yeast, host cells may over- glycosylate the gene product. When using such expression systems it may be preferable to alter CTGF coding sequence to el; ;n~te any N-l;nke~ glycosylation site.
The CTGF sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for scr~n;ng of peptide libraries it may be useful to encode a ch; ~ric CTGF protein expressing a heterologous epitope that is recognized by a l_ ?~cially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the CTGF sequence and the heterologous protein sequence (e.g. a sequence encoding a growth factor related to PDGF), so that CTGF can be cleaved away from the heterologous moiety.
The co~;ng sequence of CTGF may also be synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers, et al., 1980, Nucleic Acids Res. Symp. Ser. 7:215-233; Crea and Horn, 1980, Nucleic Acids Res. 9(10):2331; Matteucci and Caruthers, 1980, Tetrahedron Letters 21:719; and Chow and Kempe, 1981, Nucleic Acids Res. 9(12):2807-2817. Alternatively, the protein itself could be produced using chemical methods to synthesize the CTGF amino acid sequence in whole or in part. For example, peptides can be synthesized by solid phase techn;ques, cleaved from the resin, and purified by preparative high performance liquid chromatography. See e.g., Creighton, 1983, Proteins Structures And Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing. See e.g., for the Edman degradation procedure, see, Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 34-49.
A more detailed description of the nucleic acid sequences comprising the present invention and methods for identifying such sequences may be found in U.S. Patent, W O96/38168 PCTrUS96/08210 Serial No. 5,408,040, which is incorporated herein by reference.
Expre3sio~ Of CTGF. In order to express a biologically active CTGF, the nucleotide se~uence coding for the protein, or a functional equivalent as described above, supra, was inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted co~;ng sequence.
More specifically, methods which are well known to those skilled in the art can be used to construct expression vectors containing the CTGF sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA t~r-hn;ques, synthetic t~çhn;ques and in vivo recombination/genetic recombination.
See e.g., the t~chn;ques described in Maniatis et al., 1989, Mol~c~ r Cloning: A Laboratory M~nll~l, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Mol~clll~r Biology, Greene Publ;ch;ng Associates and Wiley Interscience, N.Y.
A variety of host-expression vector systems may be utilized to express the CTGF coding sequence. These include but are not limited to microorg~n; - such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA
or cosmid DNA expression vectors contA;n;ng the CTGF coding sequence; yeast, including Pichia pastoris and Hansenula polymorpha, transformed with recombinant expression vectors contA;n;ng the CTGF coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., bacculovirus) containing the CTGF coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression ~ vectors (e.g., Ti plasmid) contA;n;ng the CTGF coding sequence, or An; ~1 cell systems infected with recombinant virus expression vectors (e.g., adenovirus, vaccinia virus, human tumor cells (including HT-1080)) including cell lines engineered to contain multiple copies of the CTGF DNA either CA 02222609 1997~ 27 W O96/38168 PCTrUS96/08210 stably amplified (CHO/dhfr) or unstably amplified in double-minute chromosomes (e.g., murine cell lines). As used herein, it is understood that the term "host-expression vector systems" and more generally, the term "host cellsn includes any progeny of the host cell or host-expression vector system. It is further understood that although all progeny may not be identical to the parental cell, as mutations may occur during replication, such progeny are included in the scope of the invention.
The expression elements of these systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector.
For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage ~, plac, ptrp, ptac (ptrp-lac hybrid ~ll -Ler) and the like may be used; when cloning in insect cell systems, promoters such as the bacculovirus polyhedrin ~l~ -Ler may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the ~l~ -Ler for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA
~ ~ -Ler of CaMV; the coat protein promoter of TMV) may be used; when cloning in - -lian cell systems, promoters derived from the genome of ~ -lian cells (e.g., metallothionein ~I~ -Ler) or from r '1 ;~n viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K
promoter) may be used; when generating cell lines that contain multiple copies of the CTGF DNA SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the expressed CTGF. For example, a suitable vector for expression in bacteria includes the T7-based vector as described in Rosenberg, et al . ., 1987, Gene 56:125. As further example, when large ~uantities of CTGF are to be W O96/38168 PCT~US96108210 produced to screen peptide libraries, vectors which direct the expression of high levels of protein product. that are readily purified may be desirable. Such vectors include but are not limited to the E. coli expression vector pUR278 (Ruther et al ., 1983, EMBO J. 2:1791), in which the CTGF
coding sequence may be ligated into the vector in frame with the lac Z coding region so that a hybrid AS-lac Z protein is produced; pIN vectors (Inouye ~ Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke ~ Schuster, 1989, J. Biol . Chem.
264:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides such as CTGF with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety.
More generally, where the host is a procarote, competent cells which are capable of DNA uptake can be prepared from cells harvested after ~xronential growth and subsequently treated by the CaCl2 , or alternatively MgCl2 or RbCl, method using procedures well known in the art.
Where the host cell is a eucaryote, various methods of DNA transfer can be used. These include transfection of DNA
by calcium phosphate-precipitates, conventional -c-b~n;cal procedures, including microinjection, insertion of a plasmid encased in liposomes, or use of virus vectors. Eucaryotic cells may also be cotransformed with DNA sequences encoding the polypeptide of the invention, and a second foreign DNA
molecule encoding a selectable phenotype, such as herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eucaryotic cells and express protein. See, Eukaryotic Viral Vectors, 1992, Cold Spring Harbor Laboratory, Gluzman, Ed.).
Eucaryotic host cells include yeast, - ~lian cells, insect cells and plant cells.

W O96/38168 PCTrUS96J'B210 In yeast, a - h~r of vectors cont~; n; ng constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ausubel et al., Ed., Greene Publish. Assoc. & Wiley Interscience, Ch. 13;
Grant et al., 1987, Methods in Enzymology, Wu & Grossman, Eds., Acad. Press, N.Y., 153:516-544; Glover, 1986, DNA
Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Berger & Kimmel, Eds., Acad. Press, N.Y., 152:673--684; and The Molec~ r Biology of the Yeast Saccharomyces, 1982, Strathern et al., Eds., Cold Spring Harbor Press, Vols. I and II. For example, various shuttle vectors for the expression of foreign genes in yeast have been reported. Heinemann, et al., 1989,Nature 340:205; Rose, et al., 1987,Gene 60:237.
In cases where plant expression vectors are used, the expression of the CTGF coding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and l9S RNA ~l~ -Lers of CaMV (Brisson et al ., 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311) may be used;
alternatively, plant ~ ~ers such as the small subunit of RUBISC0 (Coruzzi et al ., 1984, EMBO ~. 3:1671-1680; Broglie et al ., 1984, science 224:838-843); or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B (Gurley et al ., 1986, Mol . Cell . Biol . 6:559-565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such t~chn;ques, see, e.g., Weissbach & Weissbach, 1988, Methods for Plant Mol~c~ r Biology, Academic Press, NY, Section VIII, pp. 421-463; Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.
In an insect system, an alternative expression system could be used to express CTGF. In one such system, Bacculovirus is used as a vector to express foreign genes.
The virus then grows in the insect cells. The CTGF coding sequence may be cloned into non-essential regions (for W O96138168 PCTrUS96/08210 example the polyhedrin gene) of the virus and placed under control of a Bacculovirus promoter. These recombinant viruses are then used to infect insect cells in which the inserted gene is expressed. See, e.g., Smith et al., 1983, ~. Virol. 46:584; Smith, U.S. Patent No. 4,215,051.
In ~ -lian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the CTGF coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This ch; eric gene may then be inserted in the adenovirus geno~e by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing CTGF in infected hosts. See e.g., Logan & Shenk, 1984, Proc.
Natl. Acad. sci. (USA) 81:3655-3659. Alternatively, the vaccinia 7.5K ~-~ ~Ler may be used. See, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci. (USA) 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931.
In another embodiment, the CTGF sequence is expressed in human tumor cells, such as HT-1080, which have been stably transfected with calcium phosphate precipitation and a neomycin resistance gene. In yet another embodiment, the pMSXND expression vector or the like is used for expression in a variety of - ~ n cells, including COS, BHK 293 and CH0 cells. Lee and Nathans, 1988, ~. Biol. Chem. 263:3521.
Specific initiation signals may also be required for efficient translation of inserted CTGF coding sequences.
These signals include the ATG initiation codon and adjacent sequences. In cases where the entire CTGF gene, including its own initiation codon and adjacent sequences, is inserted - into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the CTGF coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the W O96/38168 PCTrUS96/08210 initiation codon must be in phase with the reading frame of the CTGF coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be ~nhAnc~ by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. see e . g., Bitter et al ., 1987, Methods in Enzymol . 153:516-544.
In addition, a host cell strain may be chosen which modulates the expression of the inserted se~lences, or modifies and processes the gene product in the specific fashion desired. Such modirications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific ?c-hAn; for the post-translational processing and modification of proteins.
A~L~liate cells lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such - -lian host cells include but are not limited to CH0, VER0, BHK, HeLa, COS, MDCK, 293, WI38, HT-1080 etc.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express CTGF may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with CTGF DNA
~o.,LLolled by a~Lo~Liate expression ~oi.~Lol elements (e.g., promoter, ~nhAnc~, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
Following the i.,~Lod~ction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and ~xr~n~e~ into cell lines.
A lll he~ of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szyb~lska & Szyh~lsk;, 1962, Proc.
Natl. Acad. Sci. (USA) 48:2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt~ or aprt~ cells, respectively.
Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Proc. Natl. Acad. Sci.
(USA) 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci.
(USA) 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. sci. (USA) 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol . ~iol . 150:1);
and hygro, which confers resistance to hyyr- ~cin (Santerre, et al., 1984, Gene 30:147) genes. Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, 1988, Proc. Natl. Acad. sci. (USA) 85:8047), and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In:
Current C nications in Molecular Biology, Cold Spring Harbor Laboratory ).
The isolation and purification of host cell expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and ; ological separations such as those involving the use of monoclonal or polyclonal antibody.

7.2. Identification Of Transfectants or Transformants That Expross CTGF
The host cells which contain the coding sequence and which express the biologically active gene product may be identified by at least four general approaches: (a) DNA-DNA
or DNA-RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by the expression of CTGF mRNA
transcripts in the host cell; and (d) detection of the gene product as measured by an assay or by its biological activity.
In the first approach, the presence of the CTGF coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the CTGF coding sequence, respectively, or portions or derivatives thereof.
In the second approach, the recombinant expression vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., resistance to antibiotics, resistance to methotrexate, trans~ormation phenotype, occlusion body formation in bacculovirus, etc.). For example, in a preferred embodiment, the CTGF coding sequence is inserted within a n~_ y~in 2S resistance marker gene sequence of the vector, and recombinants con~;n;ng the CTGF coding sequence can be identified by the absence of the marker gene function.
Alternatively, a marker gene can be placed in tandem with the CTGF sequence under the control of the same or different promoter used to control the expression of the CTGF coding sequence. Expression of the marker in response to induction or selection indicates expression of the CTGF coding sequence.
In the third approach, transcriptional activity for the CTGF coding region can be assessed by hybridization assays.
For example, RNA can be isolated and analyzed by Northern blot using a probe homologous to the CTGF coding sequence or particular portions thereof. Alternatively, total nucleic W O96/38168 PCTrUS96/08210 acids of the host cell may be extracted and assayed for hybridization to such probes.
The fourth approach involves the detection of the biologically active or immunologically reactive CTGF gene product. A number of assays can be used to detect CTGF
activity including but not limited to those assays described in U.S. Patent No. 5,408,040.

7.3~. Tre~tment Indications The methods, compounds and formulations of the present invention are each directed to the treatment of disorders, diseases or ailments related to the undeL~lGd~ction of connective tissue in bone, cartilage, or other organs such as skin and muscle alternatively, to disorders, diseases or A; 1 -~tS in which the formation of bone or cartilage is desired.
These diseases, disorders or ailments include the repair of cartilage or bone defect after a variety of traumatic injuries or disorders including arthritis, osteoporosis and other skeletal disorders, hypertrophic scars, burns, and vascular hypertrophy. Because these problems are due to a poor growth response of the fibroblasts, stem cells, chondrocytes, osteoblasts or fibroblasts at the site of injury, the addition of a biologically active agent which could stimulate the growth of these cells would be beneficial.
Another important use of CTGF would be in culture systems to expand stem cells or chondrocytes that were ~ ed from an individual prior to reimplantation. In a similar process, CTGF could be added to either stem cells or Ghon~rocytes when they were to be added as a graft to help stimulate the ~xr~n~ion and differentiation of these cells at the site of implantation. CTGF could also be added to a graft composed of cartilage or bone to help stimulate growth.
Another treatment indication is directed to ~, ;n;~tering CTGF to a patient in need to enhance wound healing. PDGF and other growth factors, such as CTGF, are involved in normal healing of skin wounds. The CTGF

, polypeptide of this invention is valuable as a therapeutic in cases in which there is i ~;red healing of skin wounds or there is a need to augment the normal healing ?chAn;! , e.g., burns. One important advantage to using CTGF protein to accelerate wound h~Al ing is attributable to the molecule's high percentage of cysteine residues. CTGF, or functional fragments thereof, is more stable and less susceptible to protease degradation than PDGF and other growth factors known to be involved in wound healing.
Preferably, the agent of this invention is the combination of TGF-~ an~ CTGF, however, it is likely that other TGF-~ family members will also be useful in accelerating wound healing by inducing CTGF. The composition of the invention aids in healing the wound, in part, by promoting the growth of connective tissue. The composition is prepared by combining, in a pharmaceutically acceptable carrier substance, e.g., inert gels or licluids~ the purified CTGF and TGF-~.
The treatment indications, with respect to wound healing, contemplated by this invention include anticipated wounds (i.e. wounds resulting from surgical procedures), as well as unanticipated wounds (i.e. wounds caused by trauma).
7.4. phA ~s~tic_l Formulations And Rout~s Of ~~ ~ n ~ ~tration The molecules of the present invention can be AC' ; n; ~tered to a patient in need, by itself, or in pharmaceutical compositions where one or more of the molecules are mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders.
Alternatively, as CTGF is produced by endothelial cells and fibroblastic cells, both of which are present at the site of bone or cartilage formation and wolln~; ng, agents which stimulate the production of CTGF can be added to a composition which is used to accelerate bone or cartilage induction or wound healing. Preferably, the agent of this invention is transfo~ ; ng growth factor beta. The composition of the invention aids in healing the wound, in CA 02222609 l997-ll-27 W O96/38168 PCTrUS96/08210 part, by ~ -Ling the growth of co~nective tissue. In another embodiment, CTGF may be ~, ;n;~tered in combination with proteins or compounds which are believed to ~l~ ~Le the formation of connective tissue.
Whether the composition is comprised of CTGF alone or CTGF and additional agents as the active ingredient, such composition is prepared by combining, in a pharmaceutically acceptable carrier substance, e.g., inert gels or liquids, the purified CTGF and TGF-~.
A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms. Techniques for formulation and ~ ;stration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition.

7.4.1. Routes Of Administration.
Suitable routes of Al' ; n;~tration may, for example, include oral, rectal, trAn! cosal, or intestinal AC' ; n;~tration; parenteral delivery, including intramuscular, subcutaneous, i~Ll ~ullary injections, as well as intrath~cAl, direct intraventricular, intravenous, intraperitoneal, intrAnA~A-, or intraocular injections.
Alternately, one may A~' ; n;~ter the compound in a local rather than systemic manner, for example, via injection of the compound directly into an area requiring CTGF, often in a depot or sustA; ne~ release formulation.
Furthermore, one may a ;n;~ter the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, cartilage. The liposomes will be targeted to and taken up selectively by the afflicted tissue.

7.4.2. Composition/Fs~ l~tion.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, W O96/38168 PCTrUS96/08210 granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active mGlecules into preparations which can be used pharmaceutically. Proper fo~ lAtion is dependent upon the route of A~- ; n; stration chosen.
For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For trAn! çosal administration, penetrants appropriate to the barrier to be permeated are ussd in the foL ~lAtion. Such penetrants are generally known in the art.
For oral A, ; n; stration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
Such carriers enable the 5: po~ ds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydro~y~lo~ylmethyl-cellulose, sodium carboxy ?thylcellulose, and/or polyvinylpyrrolidone (PVP).
If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which W O96/38168 PCTrUS96108210 may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All fG~ l~tions for oral ~, ; n; ~tration should be in dosages suitable for such administration.
For buccal At' ;n; ~tration,the compositions may take the form of tablets or lozenges foL l~ted in conventional ~nner .
For ~- ;n;~tration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be detel ;ne~ by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated cont~;n;ng a powder mix of the compound and a suitable powder base such as lactose or starch.
The molecules may be fo, lAted for parenteral a~ ;n;~tration by injection, e.g., by bolus injection or continuous infusion. Fol lations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-W O96~8168 PCT~US96/08210 dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical foL 11 ~tions for parenteral ~- ;n;~tration include aqueous solutions of the active c~ ,oullds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium calbGxy ?thyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.~., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., contA;n;ng conventional suppository bases such as cocoa butter or other glycerides.
In addition to the fGl lAtions described previously, the compounds may also be formulated as a depot preparation.
Such long acting foL lAtions may be a~ ;n;~tered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be fol lAted with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion ~ch~nge resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
A pharmaceutical carrier for the hydrophobic molecules of the invention is a cosolvent system comprising benzyl W O96/38168 PCT~US96/08210 alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8~ w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself pro~llc~c low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without~destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other bisc~ pAtible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
Alternatively, other delivery systems for hydrophobic molecules may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the r~ _ounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydl~hobic polymers cont~; n; ng the therapeutic agent. Various of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over lO0 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, W O96138168 PCTrUS96/08210 starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

7.4.3. Effective Dosago.
Pharmaceutical c r,-sitions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its int~nA~A purpose. More specifically, a therapeutically effective amount means an ~ u~.~ effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be fol l~ted in ~n i ~1 models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half~ l CTGF activity). Such information can be used to more accurately determine useful doses in h - n.~ .
A therapeutically effective dose refers to that amount of the molecule hat results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such molecules can be determined by stAnA~d pharmaceutical procedures in cell cultures or experimental ~n; -lS, e.g., for detel ;n;ng the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Molecules which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and ~n; ~1 studies can be used in fo~ l~ting a range of dosage for use in human. The dosage of such molecules lies preferably within a range of W O96/38168 PCTrUS96108210 circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of A~' ;n;~tration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l.
Dosage A ullL and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the induction effects of CTGF, or ;n; ~1 effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; for example, the concentration nece~sary to achieve 50-90%
activity of CTGF to induce bone growth using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using MEC value.
Compounds should be A~' ; n; ~tered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
In cases of local administration or selective uptake, the effective local rs~c~ntration of the drug may not be related to plasma concentration.
The A -ullL of composition a~~ ;n;~tered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of AS ; n;~tration and the jll~_ -nt of the prescribing physician.

7.4.4. Packaging The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms contA;n;ng the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be acc~ _~n;ed by instructions for A-- ;n;~tration~ Compositions W O96/38168 PCTrUS96108210 comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate cont~;n~ and labelled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of disorders or diseases in which cartilage or bone induction and wound healing, or the like is desired.

7.5 Identification of Compounds Which Induce The Production of CTGF In Cartilage And The identification of the promoter element of the CTGF gene and specifically, the TGF-~ responsive/regulatory element (T~RE) (5'-GTGTCAACCCTC-3~; nucleotides -157 and -145), provides a source for a scr~;nq method for identifying compounds or compositions which affect the expression of CTGF. Specifically, the method by which compositions which ~nh~nc~ the activity of CTGF, and thereby may be used to ~nhAnc~ bone, tissue and cartilage induction may be identified, comprises: (1) insllh~ting components such as, but not limited to, oligonucleotides comprising the composition and a TGF-~ responsive element of the CTGF
promoter, wherein said inr~lh~tion is carried out under conditions sufficient to allow the r~ onents to interact;
and (2) measuring the effect of the c~ _ -ition on CTGF
expression. Preferably, the promoter region used in the screening assays described herein includes nucleotides -823 to +74, although smaller regions that include the TGF-~
responsive element may also be useful in the disclosed method (e.g. -162 to -128, or -154 to -145). In other assays, nucleotides in this region including T~RE are coupled to a receptor gene such as luciferase and are transfected into - -lian cells to derive a cell line, bearing a construct and showing activity when incubated with TGF-~. These drugs, oligonucleotides, and chemicals in libraries that modify the activation can be readily detected in cell assays.

W 096/38168 pcTrus96lo82lo 8. Eau~MPLE8 The connective tissue growth factor gene is not only expressed in fibroblasts but is selectively induced by TGF-~ only in mesenchymally derived connective tissue cells (i.e., fibroblasts, smooth muscle, chondrocytes, osteoblasts, astroglial cells, etc.). The expression of the gene connective tissue cells that form the skeletal elements in vertebrates indicate that CTGF plays a role in the formation of cartilage, bone tendon and muscle in the vertebrate ~n; ~1. The results of the following examples demonstrate that CTGF can regulate the induction, differentiation and growth of cells which form both cartilage and bone in vertebrate ~n;~ , including humans. Specifically, the results provide that: (1) CTGF transcripts are present in the growth plate of long bones in adult rats and newborn mice;
(2) The CTGF gene is expressed ~t sites of cartilage induction and growth in embryonic mice; (3) CTGF receptors are present on rat chondrocytes; (4) The CTGF gene is expressed at site of bone regeneration after injury in adult rabbits; (5) The CTGF protein can ;n~nc~ pluripotent mouse embryonic stem cell lines to differentiate in to chondrocytes and osteoblasts; 6) Human osteoblasts produce CTGF in culture.

8.1 Biologic~l Ass~ys Methods: Mitogenic And An~ho~age Inder~nt Growth A~says. Mitogenic assays were performed in monolayer cultures using 48 well plates and NRK fibroblasts as target cells as described previously in Grot~n~orst, et al. 1991, J.
Cell Physiol. 149:235-243. Anchorage independent growth assays were performed essentially as described in Guadagno and Assoian, 1991, J. Cell Biol. 115:1572-1575.
Methods: Extracellular Matrix Protein mRNA
Induction A3says. NRK rat fibroblasts were grown to confluence in Dlllh~cco's modified eagle media with 5% gfetal bovine serum and then serum starved in DMEM with 1% bovine serum albumin for 24 hours. Growth factors were added to the cell cultures and total cellular RNA was extracted after 24 W O96138168 PCTrUS96/08210 hours and northern blot analysis was performed as described in Igarashi, et al ., 1993, Mol . Biol . Cell 4:637-645.
To ensure that equivalent amounts of total RNA were added to each lane on a gel, RNA was quantitated by A260~280 ratios and equivalent transfer was assured by c_ -ring ribosomal 28S and 18S RNA bands in each lane after st~;n;ng with ethidium bromide. As additional control, blots were reprobed with an actin cDNA probe. Double stranded cDNA
fragments used for probes were labeled with 32P-dCTP using a random prime l~h~l ;ng kit (Boehringer M~nnh~im, Tn~;An~polis, IN). The CTGF probe was derived from a 1.1 kb human cDNA
fragment which enc~ -ssed the open reading frame of the CTGF
transcript. The TGF-~1 probe was a 1.0 kb Nar I fragment derived from a 2.0 kb human TGF-~1 cDNA (G.I. Bell, H.H.
Medical Institute, University of Chicago). The ~I-type 1 human collagen probe was derived from a 1.5 kb ORF fragment at the 3'end (ATCC No. 61323). The ~5 integrin probe was produced from a cDNA insert cont~;n;ng a portion of the human cDNA cont~;n;ng the open r~; ng frame, as obtained by R.
Associan at the University of Miami. The human fibronectin probe was a O.9 kb EcoR1/HindIII fragment derived from a 2.2 kb cDNA clone cont~; n; ng the 3' region of the open reading frame provided by F. Woessner (also of University of Miami).
The human actin probe, used as the control RNA probe, was purchased from Oncor, Co. (Gaithersberg, MD).

8.2 Locus Of CTGF Tr~n3cripts In Newborn ~ico Experiments were conducted to determine whether CTGF transcripts are present in the growth plate of long bones of newborn mice according to Fava, et al., 1990, Blood 76:1946-1955.
Method: In RitU Hybridization. The tissue samples were ; ~ tely placed in 4.0% paraformaldehyde for 1.5 hours and then flash frozen and h~e~. Sections were cut at 5 ~m and placed on TESPA coated slides (Oncor, Gathersburg, MD). In-situ hybridization for CTGF mRNA was performed using st~n~rd methods. Briefly, slides with specimens were hydrated through graded alcohols, treated with 20 ~g/ml proteinase K in 50 mM Tris-HCl ph 7.4, 5 mM EDTA, refixed in 4.0% paraformaldehyde and dipped in 0.1 M
triethanolamine and 1 ml acetic anhydride, prior to dehydration in sequentially graded alcohols. Both sense and antisense CTGF RNA probes were constructed using a riboprobe kit (Plc ?ga, Madison, WI) with T7 and Sp6 promoters, respectively. The specific activity of the probes was 1 x 108 cpm/~g RNA. Slides were hybridized overnight in 50%
deionized fo - ;de, 10% dextran sulfate, 50 mM DTT, 0.3M
NaCl, 0.01 M Tris pH 7.5, 5 mM EDTA, 10 mM Na2HP04, 0.02 %
Ficoll, 0.02 % PVP, 0.02 % BSA, 0.2 mg/ml yeast tRNA and the riboprobe ( 5 x 104 cpm/~l) under a coverslip at 54~ C.
Slides were washed in 250 ml 5X SSC, 10 mM beta mercaptoethanol at 50~ C for 30 minutes, 2x SSC, lOOmM beta mercaptoethanol, 50% formamide at 65~ C for 20 minutes and 3 times in TEN buffer (1 M Tris, 0.5 M EDTA, 5M NaCl) for 10 minutes. The second TEN wash included 10 ~g RNase A. The final two washes were in 2X SSC at 65~ C. for 15 minutes each. After dehydrating again through graded alcohols with 0.3 M ammonium acetate, the slides were dipped in photographic emulsion (Ilford K-5, Polyscience) and incubated for 8 days at 4~ C. Slides were then developed and sections counter st~; n~ in Mayer's hematoxylin and eosin.
Resul t~ . The results of these studies indicate that the CTGF gene is expressed in the proliferation zone of the growth plate. This zone contains the chrondrocytes that are actively replicating to increase the length of the bone.
The expression of CTGF at this site is consistent with it functioning as a growth factor for the chondrocytes.

8.3 CTGF Gene ~ ~Osion At 8ite of C~rtilage Induction And Growth In Embryonic Mice.
In order to confirm CTGFs role in cartilage induction and growth, the expression of the CTGF gene in mouse embryo's at sites where cartilage and bone will form but have not formed yet was studied. For purposes of this study, a transgenic mouse line which contains a transgene composed of the human CTGF promoter elements which are W O96/38168 PCTrUS96/08210 regulating the expression of the bacterial ~-galactosidase gene. Cells that express this gene can be readily identified by st~;n;ng using X-gal which forms a blue colored precipitate at the sites of enzymatic activity. Using this methodology we stained embryo's from transgenic mice to localize the expression of the CTGF gene. As set forth at FIGURE 4, Panel A, no cartilage or bone was formed, indicating the CTGF is expressed prior to the formation of the skeleton and could function as the inducer of cartilage and bone. These results further demonstrate that the expression of the transgene corresponds with the expression detected by in situ hybridization using the CTGF probe.
These studies also demonstrate that the gene is expressed at growth plates in the long bone, in precartilaginous zones and in M~ckel's cartilage, the first cartilage to form during ~ development. These areas are referred to as pr~hon~ogenic mesenchyme and are distingn;~he~ by condensations of the cells. The CTGF gene is expressed in these sites but not in adjacent tissue.
Furthermore, the CTGF gene is expressed at these sites 1 day prior to the condensation which occurs 1 day prior to the actual formation of the cartilage. These f;n~; ngS
demonstrate that CTGF is present prior to the formation of cartilage or cells with a true chondrocytic phenotype, and is consistent with CTGF acting to ;n~llr~ the cartilage phenotype in undifferentiated stem cells.
Importantly, these studies demonstrate that CTGF is expressed at sites in the embryo that form bone by either the intr~ --h~anous, or endochondral pathways, demonstrating that it can function as a signal for cartilage development from either non-differentiated mesenchymal stem cells which form the bones of the limbs, or neural crest cells which form the cartilage in Meckel's cartilage and the bones of the skull.

8.4 Loci Of CTGF Receptors On R~t Chondrocytes In order for cells to respond to peptide factors such as CTGF, they must express on their surface the cognate receptor for the specific peptide factor.

W O96/38168 PCTrUS96/08210 Equil ibrium Ass~y. Equilibrium binding assays were performed on confluent monolayers of NRK-49F rat fibroblasts and primary rat articular chondroblasts to determine the number and affinity of CTGF receptors on these cells.
Binding was performed in the cold for 4 hours with varying co~c~ntrations of iodinated recombinant human CTGF (rhCTGF).
Non-specific b;n~;ng was detel ;n~ by including a 200-fold molar excess of unlabeled ligand. Representative Scatchard plots are set forth at FIGURE 8A (with respect to equilibrium binding assays perfomed using NRK cells) and FIGURE 8B (with respect to equilibrium binding assays performed using rat chondroblasts).
Competition Ass~y. Several cell types were tested for the expression of CTGF receptors including, normal rat lS kidney fibroblasts, mouse fibroblasts, mink lung epithelial cells and rat articular rho~ocytes. CTGF was labeled by iodination with l25I and the radiolabled CTGF used in competition b; n~; ng assays to measure CTGF receptors on the various cell types. As set forth in Table 1, below, only the NRK fibroblasts and rat articular ~hon~rocytes expressed high affinity receptors for CTGF. Mouse fibroblasts had few if any high affinity receptors and no binding was detected in the mink lung epithelial cells.
TABLE
BINDING CH~RACTERISTIC FOR rhCTGF ON VARIOUS CELLS
His~h Affinitv Low Affinitv Cell Tvpe KD (pM) Sites/cell RD (nM) Sites/cell NRR 13-23 Z200-3500 1.1-2.2 126,000-195,000 3S Chon.l,u~ y-~ Z1 3500-4800 1.0 150,000 NIH3T3 5-10 480 1.8 102,000 M~EC none detected none detected These data indicate that cho~ocytes express both CTGF
and its receptor and would are therefore capable of responding to CTGF as a growth stimulatory factor.

8.5 CTGF Activity In Inducing Pluripotent Nouse Embryonic Stem Cell Lines To Differentiate Into ~hQn~rocytes And Osteoblasts-The ability of CTGF to ;n~tlc~ the rho~rocytic and osteocytic phenotype in undifferentiated stem cells in cell culture was evaluated. Specifically, the cell line C3HlOT1/2 was used to evaluate this biological activity. These cells are a st~n~rd and well established line for these types of investigations. The C3HlOT1/2 can be maint~;ne~ in an undifferentiated state in culture, and then induced to differentiate into skeletal muscle cells, chondrocytes, osteoblasts and adipocytes. Cells treated with CTGF formed colonies of chon~ocytes and cartilaginous nodules. Cells treated overnight with 5-azacyto~;ne overnight followed by treatment with CTGF differentiated into osteoblasts and osteoid bodies. The differentiation of these cultures into muscle and adipocytes was blocked by the presence of CTGF.
More specifically, the cells were treated overnight with 5 azacytodine followed by a 10-14 day ;nrllh~tion to allow for the differentiation to occur. The effects of 5 azacytodine and CTGF alone and in combination on these cell were then r_ -red Control cultures which were not treated with either agent r~ -; n~ as undifferentiated cells in monolayer.
As set forth as FIGURE 4, cultures treated with 5azacytodine alone differentiated into primarily skeletal muscle cells (myotubes) and adipocytes. No chondrocytes could be found in the cultures. CTGF treatment of the cultures (50 ng/ml) for 10 days resulted in the induction of cartilaginous nodules.
These nodules were not found under any other conditions.
Treatment of the cultures with FGF, PDGF, EGF or TGF-~ did not induce these nodules indicating that CTGF is uniquely capable of inducing cartilage in the undifferentiated mesenchymal stem cells.
Treatment of the cells with both 5 azacyto~; ne (overnight) followed by a 10 day exposure to CTGF (50 ng/ml) had a significant effect on the cultures. First, no skeletal myotubes where present demonstrating that CTGF prevented the cells from differentiating into skeletal muscle cells.

W O96/38168 PCTrUS9~ .10 Second, while some cartilaginous nodules were present most of the nodules appeared to be osteoid (bone). Thus, CTGF can induce the formation of both chondrocytes and osteoblasts from undifferentiated mesenchymal cells. These results ~ -ctrate that this factor could be used to st; lAte the differentiation of cartilage and bone where desired.
8.6 CTGF Gene ~A~.~3sion At 8ite Of Bone Regenoration After Injury In Adult Rabbits.
An experimental model was developed to ~ ;ne the expression of various regulatory and matrix protein genes during wound repair. In this model, mesh nylon cyl;n~e~s are implanted in the ilium of the pelvis of male New Z~A l An~
white rabbits (10 kg) which had been anesthetized by ether.
A 1.1 cm diameter hole was bored in the ilium of the pelvis using a bone tr~ph; ne and the chamber press fit into the hole. The ~~hA h~r was An~hol~ed in place using flaps of the adjacent musculature and ligaments. Two chambers were implanted in each of twenty An; -lc.
~n; -l~ were sacrificed on Days 9, 14, 21, 24, 28, 31, 35, 42 and 56 after implantation of the ch; h~rs. The chambers were removed and the tissue on the outside of the ~hA h~s were carefully and completely ~ ved. The ~hA h~s were then cut open and tissue contained within the ~-hA h~s was collected.
Total RNA was extracted from the tissue obtained from 6-18 chambers (pooled from 1-3 An; ~l ~) by Guanidine-isothiocynate extraction (Chomcaynski and Sacchi, 1987, Anal.
Biochem. 162:156-159) and CsCl centrifugation (Chirgwin, et al., 1979, Bioche ictry I8:5294-5299). The amount of RNA
recovered ranged from 100-300~g during the different days of collection. Total RNA was electrophoresed on an agarose/formaldehyde gel and transferred to nitrocellulose.
Equivalent amounts of RNA were transferred as judged from st~; n; ng of the ribosomal RNA present in each sample on the nitroceIlulose filter. The CTGF probe was a 900 base pair fragment which represented the open reading frame of the CTGF
cDNA. Hybridizations were performed using 1x106 cpm/ml of W O96/38168 PCTrUS9~ 10 these probes labeled with ~P32]dCTP by using a RAn~, Primer DNA Labeling Kit (Boehringer Mannheim Biochemicals, Tn~;~n~rolis, IN). Autoradiography was performed at -70~C
for 24-72 hours by using X-ray films and intensifying screens.
The tissue proceeded through a regular cascade of repair where blood coagulation is followed by inflammation and then connective tissue in growth. In the bone implanted chambers the dense connective tissue which formed was similar if not indistingll;~h~hle from that which forms in soft tissue implants.
As set forth in FIGURE 5, CTGF gene expression is evident from Days 14-42. This is 4 days prior to the first histological appearance of bone within the c-h;~ h~s and coincides with the time course for the formation of bone within the t-h;~ h~rs. However, as also set forth at FIGURE 5, around day 17-18 post-implantation there were some changes in the morphology of areas of the connective tissue. These areas then began to form bone by day 20-21 post-implantation, demonstrating that this is a functional model for the study of bone regeneration.
The expression of CTGF mRNA in the chambers pr~c~e~
slightly and then coincided with the formation and growth of the osteogenic areas within the ~h~ h~r, ~ n~:trating that CTGF is expressed at sites of bone regeneration in 8.7 ~um~n Osteoblast Formation By ~ tration 0 CTGF
Cell cul ture . Human osteoblasts were grown from explants of human bone. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) cont~;n;ng 10 % fetal calf serum (FCS) at 37 C in an atmosphere of 10 % C02 and 90 %
air.
Western blot analysis. CTGF content in conditioned media was analyzed by SDS-PAGE on 12 % acrylamide gels followed by transfer to nitrocellulose filters using electroblotting. The blots were ;ncllh~ted for 1 hour in Tris-buffered saline (lOOmM NaCl, 50mM Tris-HCl pH7.4) with 2 W O96/38168 PCTrUS96/08210 % nonfat powdered milk (TBS-milk), prior to overnight exposure to 2 g/ml chicken anti-human CTGF IgY diluted in TBS-milk. Filters were washed five times in TBS-milk, 5 minutes each, and ;ncnh~ted with alkaline-phosphatase-conjugated affinity purified rabbit anti-chicken IgY (1:1,000 dilution, Organon Teknika-Cappel, West Chester, Pa.) in TBS-milk for 90 minutes. The filters were washed three times with TBS-milk followed by two washes in TBS, and the antigens were detected using a commercial alkaline phosphatase substrate kit (Sigma, St. Louis, MO).
Results. Human osteoblasts were obt~;ne~ from donors after surgical removal of bone during procedures to remove bone tumors or joint replacement. Osteoblasts were cultured from the bone and identified using st~n~d. Cells were grown to confluence in complete media cont~;n;ng 10 %
fetal calf serum and made quiescent by changing the media to serum free media overnight. Some cultures were treated with TGF-~ and compared to non-treated cultures. The osteoblast that were treated with TGF-~ were stimulated to produce CTGF, as detected with a specific anti-CTGF antibody. As set forth at FIGURE 5, the media was collected and analyzed for CTGF
production and secretion by ; opurification of the CTGF
with a CTGF specific antibody and detection and quantitation by western blots using the same antibody. As observed with fibroblasts, smooth muscle cells, and chondrocytes, TGF-~induces CTGF production by the human osteoblasts. Control non-treated cells did not synthesis detectable amounts of CTGF.
As evidenced by this experiment, osteoblasts respond to TGF-~ similarly to other connective tissue cells with regard to CTGF production.

8.8 Tr~nsgenic R~bbit M~el~
All mice studies were conducted in accordance with the principles and procedures outlined in "Guidelines for Care and Use of Experimental An; ~1~". Generation of transgenic models was carried out at the University of Miami Transgenic Mouse Core Facility using st~n~d t~chn;ques.

W O96138168 PCTrUS96/08210 Briefly, the gene to be injected (transgene) was linearized by restriction digestion and the DNA fragment isolated by low melt agarose gel electrophoresis and purified using GENECLEAN.
Transgenic mice were generated by injecting linearized DNA into one of the pronuclei of -100-300 recently fertilized mouse ova. Hogan, et al.., 1986, Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Those eggs that survive injection were transferred to the oviducts of pseudo~leyllant mice (mated to vasectomized males). One to three weeks after birth a tail biopsy will be taken from the pups and genomic DNA analyzed by southern blot to determine the presence of the transgene.
Mice that were positive for the presence of the transgene were mated to control mice to establish transgenic mouse lines. As a result of these experiments, two independent lines of transgenic mice that express the ~-galactosidase under CTGF promoter ~ollLlol were pro~lce~. Both of these lines exhibit s; ;l~ patterns of expression.
8.9 ~h~n~-ogenic Assay CTGF, as well as TGF-~ were tested in a chondrogenic assay as described in Seydin, et al., 1983, J.
Cell Biology 97:1950-53. Briefly, primary cultures of embryonic muscle were ob~in~ from cellular outgrowth of ;nc~ muscle tissue ~;cc~cted from limbs of 19-20 day old Sprague-Dawley fetuses. For the chon~ogenic assay, the cells were trypsinized and embedded in agarose, and overlayed with media cont~;n;ng no factors (FIGURE 7A), TGF-~ alone (FIGURE 7B), TGF-~ and cholera toxin (FIGURE 7C) or TGF-~, cholera toxin and CTGF (Figure 7D). For each assay, media were changed every 2 - 3 days and after 21 days of culture stained with Toluidine blue as described in Horwitz and Dorfman, 1970, J. Cell Biol. 45:434-438.
As set forth in FIGURES 7A-7D, marked ~hon~ocyte growth was observed where CTGF was added to media, indicating that CTGF st; l~tes chondrocyte growth, and the production of connective tissue matrix.

W O96/38168 PCTrUS96/08210 The present invention is not to be limited in scope by the exemplified embodiments which are inten~ as illustrations of single aspects of the invention, and methods which are functionally equivalent are within the scope of the invention. Tn~e~, various modifications of the invention in addition to those described herein will h~c- ? apparent to those skilled in the art from the foregoing description and acc _-nying drawings. Such modifications are intended to fall within the scope of the appended claims.
All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.
Bioloqical De~osits The sequence of the CTGF of the invention was deposited with Genebank, Los Alamos National Laboratory, Los Al. ~s, New Mexico, 87545, USA, on July 26, 1990, and given an accession no. M36965. The deposit of this CTFG sequence is for exemplary purposes only, and should not be taken as an admission by the Applicant that such deposit is necessary for enablement of the cl~ subject matter.
In respect of all designated States in which such action is possible and to the extent that it is legally permissible under the law of the designated State, it is requested that a sample of the deposited micro-organism be made available only by the issue thereof to an independent expert, in accordance with the relevant patent legislation, e.g., EPC rule 28(4), United Kingdom Patent Rules 1982 rule 17(3), Australian Regulation 3.25(3) and generally similar provisions mutatis mut~n~ic for any other designated State.

CA 02222609 1997~ 27 W O96/38168 PCTrUS96/08210 T ' ~ . No:PCT/
MICROORGANISMS
Option~l Shoot in connrlction with the,; ,vv, referrcd to on page 43 lines 1 2~ of the description A IDENTIFICATION OF DEPOSIT
Further deposits are idontified on an edditiomll sheet Name of depositary insdrurion GeneB~nk r~ -Address of d~ y institution (including postal code and country) Lo5 Abmo5 N~t~on~
Los Abmos, NM 87645 US
Date of deposit JulV 26, 1990 Acccssion Number M36965 B ADCIl I r.~ INDICATIONS ae ve biu~ if rnc ~pplir~ble) Thi~ infe~on i, co i~ed on ~ ~e ~ bed llee C DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE r~

D SEPARATE Fl OF INDICATIONS ae ve bbn~ irro~ ~pplic ble) Tho indication~ t d b-low wdl b- ~ubmin d to th~ Int~rnational Bur-au i-ter ISpocify th~ o-n r-l natur o~ th- indic tion- ~ o Acc~ion Nurnb-r of o-~it l E X This sheet was recei~ed with the ' applicario~ when filed (to be checl~ed by the receb~ing Office) r, ) ~zD~
(Autb~d Officer) The date of receipt (from the applicant) by the I Bureau was (Authorized Officer) Form PCTIRO/134 lJ~mu8ry 1981) _ 43.1 -

Claims (19)

WHAT IS CLAIMED

1. A pharmaceutical composition comprising CTGF.

2. A method for inducing bone formation comprising the administration to a patient in need a composition comprising CTGF and a pharmaceutical acceptable carrier.

3. The method of claim 2 wherein said composition further comprises a second growth factor.

4. The method of claim 3 wherein the second growth factor is TGF-.beta..

5. The method of claim 2 wherein said composition is further comprised of at least one collagen.

6. The method of claim 2 wherein the patient is suffering from an affliction which affects bone formation.

7. The method of claim 6 wherein the affliction is selected from the group consisting of osteoporosis, osteoarthritis and osteochondrytis.

8. A method for inducing tissue formation comprising the administration to a patient in need a composition comprising CTGF and a pharmaceutical acceptable carrier.

9. The method of claim 8 wherein said composition further comprises a second growth factor.

10. The method of claim 8 wherein the second growth factor is TGF-.beta..

11. The method of claim 8 wherein said composition is further comprised of at least one collagen.

12. A method for inducing cartilage formation comprising the administration to a patient in need a composition comprising CTGF and a pharmaceutical acceptable carrier.

13. The method of claim 12 wherein said composition further comprises a second growth factor.

14. The method of claim 13 wherein the second growth factor is TGF-.beta..

15. The method of claim 12 wherein said composition is further comprised of at least one collagen.

16. A method for inducing wound healing comprising the administration to a patient in need a composition comprising CTGF and a pharmaceutical acceptable carrier.

17. The method of claim 16 wherein said composition further comprises a second growth factor.

18. The method of claim 16 wherein the second growth factor is TGF-.beta..

19. The method of claim 16 wherein said composition is further comprised of at least one collagen.