MXPA01005948A - Connective tissue growth factor fragments and methods and uses thereof - Google Patents

Abstract

The present invention is directed to CTGF fragments comprising at least exon 2 or exon 3 of CTGF and having the ability to induce extracellular matrix synthesis, in particular, collagen synthesis and myofibroblast differentiation. The present invention is further directed to methods using said CTGF fragments to identify compositions which modulate the activity of said CTGF fragments and to the compositions so identified. The invention also relates to methods of treating CTGF-associated disorders and diseases associated with the overproduction of the extracellular matrix.

Description

FRAGMENTS OF FACTOR OF GROWTH OF CONNECTIVE TISSUE AND METHODS AND USES OF THEM F Field of the Invention This invention relates generally to the field of growth factors, and specifically to fragments of the Factor of Growth of Connective Tissue (CTGF) and methods and uses thereof. Background of the Invention Growth Factors. Growth factors can be broadly defined as multi-functional, locally acting signaling polypeptides that control both ontogeny and the maintenance of tissue form and function. The protein products of many proto-oncogenes have been identified as growth factors and growth factor receptors. Growth factors generally stimulate the target cells to proliferate, differentiate, and organize in developing tissues. The action of growth factors is dependent on their binding to specific receptors that stimulate a signaling event inside the cell. Examples of growth factors include platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), beta-transforming growth factor (TGF-β), alpha-transforming growth factor ( TGF-a), the epidermal growth factor ^ (EGF), and connective tissue growth factor (CTGF). It has been reported that each of these growth factors stimulates the cells to proliferate. Factor of Growth of Connective Tissue. CTGF is a monomeric peptide rich in cysteine with a molecular weight of approximately 38 kd. As previously reported, CTGF has both mitogenic and chemotactic activities for connective tissue cells. CTGF is secreted by cells and is believed to be active after interaction with a specific cell receptor. CTGF is a member of a family of growth regulators that includes, for example, mouse CTGF (fisp-12) and human CTGF, Cyr61 (mouse), Cef10 (chicken), and Nov (chicken). Based on sequence comparisons, it has been suggested that members of this family have a modular structure that typically consists of at least one of the following: (1) an insulin-like growth factor domain, responsible for binding; (2) a von Illebrand factor domain, responsible for complex formation; (3) a repeating troponin bospondin type I, possibly responsible for the fixation of matrix molecules; and (4) a module with terminal C that is found in the matrix proteins, postulated to be responsible for receptor binding.

The cDNA sequence for human CTGF contains an open reading frame for 1047 nucleotides, with an initiation site at approximately nucleotide 130, and a TGA termination site at approximately nucleotide 1177, and encodes a 349 amino acid peptide. The cDNA sequence for human CTGF has been previously described in U.S. Patent No. 5,408,040. The open reading frame of CTGF encodes a polypeptide containing 39 cysteine residues, indicating a protein with multiple intra-molecular bisulfide bonds. The amino terminus of the peptide contains a hydrophobic signal sequence, indicative of a secreted protein, and there are two glycosylation sites linked to N, at the asparagine 28 and 225 residues in the amino acid sequence. It is believed that the synthesis and secretion of CTGF are selectively induced by means of TGF-β and BMP-2, as well as potentially by means of other members of the super family of TGF-β proteins. As reported in the art, although TGF-β can stimulate the growth of normal fibroblasts in soft agar, CTGF alone can not induce this property in fibroblasts. However, it has been shown that the synthesis and action of CTGF are essential for TGF-β, to stimulate the growth of anchorage-independent fibroblasts. (See, for example, Kothapalli et al., 1997, Cell Growth &; Differentation 8 (1): 61-68, and Boes et al., 1999, Endocrinology 140 (4): 1575-1580). With respect to biological activity, T * has been reported that CTGF is mainly mitogenic in nature (capable of stimulating the target cells to proliferate). It has also been reported that CTGF has chemotactic activity. Pathologically, it has been reported that the full-length CTGF molecule is enveloped in conditions where there is an overgrowth of connective tissue cells, and an over-deposition of the extra-cellular matrix. It has also been described in the art that CTGF is associated with conditions related to the migration and proliferation of vascular endothelial cells, and neovascularization. Diseases and disorders related to these conditions include, for example, skin and major organ fibrosis, cancer, and related diseases and disorders such as systemic sclerosis, angiogenesis, atherosclerosis, diabetic nephropathy, and renal hypertension. (See, for example, Toshifumi et al., 1999, Journal of Cellular Physiology 1 (191): 153-159; Shimo et al., 1999, Journal of Biochemistry 126 (1): 137-145; Murphy et al., 1999, Journal of Biological Chemistry 274 (9): 5830-5834, Wenger et al., 1999, Oncogene 18 (4): 1073-1080, Frzier et al., 1997, International Journal of Biochemistry &Cell Biology 29 (1): 153- 161; Oemar et al., 1997, Circulation 95 (4): 831-839). It has also been reported that CTGF is useful in wound healing and repair of connective tissue, bone and cartilage. In this regard, CTGF has been described as Tg inducer of the formation of bone, tissue, or cartilage, in disorders such as osteoporosis, osteoarthritis, or osteochondritis, arthritis, skeletal disorders, hypertrophic scars, burns, vascular hypertrophy or healing. deep See, for example, U.S. Patent No. 5,837,258; Ohnishi et al., 1998, Journal of Molecular and Cellular Cardiology 30 (11): 2411-2422; Nakanishi et al., 1997, Biochemical and Bi ophysical Research Communications 234 (1): 206-210; Pawar et al., 1995, Journal of Cellular Physiology 165 (3): 556-565. In summary, CTGF has been implicated in numerous fibrotic and cancerous conditions, and has been described as contributing to wound healing. As a result, there is a need in the art to identify useful methods for modulating the activity of CTGF, to treat different diseases and conditions. Prior to the present invention, there has been no report that CTGF regions or domains are responsible for the signaling of different biological activities. On the other hand, prior to the present invention, there has been no description for treating diseases and disorders associated with cell proliferation and / or overproduction of extracellular matrix, by inhibiting the biological activity of a specific region or domain. of the CTGF.

SUMMARY OF THE INVENTION The present invention provides novel compositions and methods for the treatment of diseases, disorders or conditions associated with CTGF, where deposition of the extracellular matrix is involved, including, for example, the induction of Collagen synthesis and myo-fibroblast differentiation. More specifically, the compositions of the present invention comprise fragments of CTGF that comprise the N-terminal region of CTGF. In one aspect, the fragment of the present invention comprising at least a portion of exons 2 or 3, or the polypeptide encoded therein, is not the CTGF fragment disclosed by Brigstock et al., 1997, J. Bi ol. Chem. 272 (32): 20275-82, and further possesses the ability to induce extracellular matrix synthesis, including but not limited to the ability to induce collagen, and differentiation of myo-fibroblasts. In a further aspect, the fragment of the present invention comprises between about a quarter and about one half the length of the full-length CTGF protein. In one aspect, a fragment of the connective tissue growth factor (CTGF) polypeptide having the activities described above is provided. A fragment of the invention includes CTGF having an amino acid sequence encoded by at least exon 2, as indicated in Figure 3. A fragment may also include an amino acid sequence encoded by at least exon 3, as set forth in Figure 3. In addition, a CTGF fragment of the invention can include a sequence of p > amino acids encoded by at least exons 2 and 3, as indicated in Figure 3. The invention also provides sequences of polynucleotides that encode such fragments. The present invention further comprises methods of using the CTGF fragments of the disclosed invention to identify compositions that can modulate the activity of said CTGF fragments, wherein such compositions can be used to control the normal deposition of the extra-cellular matrix, such as deposition of collagen, as desired. More specifically, CTGF fragments can be used to identify compositions that can control normal collagen deposition and myo-fibroblast differentiation, where such a composition can be used to inhibit, suppress or increase the activity of CTGF fragments of the present invention. The compositions of the claimed invention further comprise CTGF modulators, for example anti-bodies, anti-sense molecules, small molecules, and other compounds identified by the above methods, which can modulate the activity of the CTGF fragments of the present invention. In one aspect, the present invention provides CTGF modulators that inhibit or suppress CTGF activity or CTGF fragments. In another aspect of the present invention, CTGF modulators increase the activity of CTGF or CTGF fragments, for example, in indications where the induction of CTGF activity is desirable, for example in wound healing, repair (jr of tissues, and bone repair In another aspect of the invention, the methods of the present invention comprise the administration of an effective amount of CTGF fragment modulators, alone or in combination with one or more compounds, to a patient who requires the treatment of diseases, disorders or conditions, where the manipulation of collagen synthesis is desired.Most particularly, the methods of the present invention are directed to using the compounds capable of modulating the activity of the CTGF fragments of this invention to modulate the synthesis of collagen, and consequently to treat disorders related to over-abundance of the synthesis of collagen, including fibrotic disorders. Preferably, the disorders are from the dermis, the major organs and disorders related to over-production of scar tissue.

The present invention also provides pharmaceutical compositions comprising the CTGF fragments of the present invention. Such compositions may be useful in wound healing, bone and tissue repair, where increased CTGF activity is desirable. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows cells in an myoblast induction assay.

Figures 2A and 2B show a graph indicating that the CTGF fragments of the present invention stimulate the T synthesis of collagen in NRK cells. Figure 3A and 3B establish the nucleic acid sequence (SEQ ID N0: 1), and the amino acid sequence (SEQ ID NO: 2) of the full-length CTGF molecule, where the location of the exons of the CTGF molecule is identified. Figure 4 establishes the nucleic acid sequence (SEQ ID NO: 3), and the amino acid sequence (SEQ ID NOM) of the N-terminal domain of CTGF, comprising exons 2 and the CTGF molecule. Figure 5 establishes the data related to the inhibition of collagen synthesis with anti-CTGF anti-bodies. Figure 6 establishes the data related to the stimulation of collagen synthesis by anti-bodies directed to the N-terminal domain of CTGF. Figure 7 establishes the data related to the stimulation of collagen synthesis by CTGF and the N-terminal domain of CTGF. Figure 8 establishes data related to the domain N-terminal CTGF as an active inducer of collagen synthesis and induction of myo-fibroblasts. Figure 9 establishes data related to the effects of IGF-2 on the synthesis of collagen induced by CTGF. Detailed Description of the Invention It is understood that the present invention is not limited to the particular methodology, protocols, cell lines, vectors, and reagents, etc. described herein, since these may vary. It will also be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It should be noted that, as used herein and in the appended claims, the singular forms "a", "an" and "the" include plural reference, unless the context clearly dictates otherwise. Thus, for example, a reference to "an anti-body" is a reference to one or more anti-bodies and equivalents thereof, known to those skilled in the art, and so on. Unless defined otherwise, all technical and scientific terms used herein have the same meanings commonly understood by one of ordinary experience in the subject matter to which this invention pertains. Preferred methods, devices, and materials are disclosed, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All references cited herein are incorporated by reference herein in their entirety. Definitions As used herein, the term "CTGF fragment" refers to a fragment comprising at least part f ^ of the N-terminal region of the CTGF. In one embodiment, the fragment comprises at least a portion of exons 2 or 3 of the full-length CTGF protein, and furthermore has the ability to induce the synthesis of the extra-cellular matrix. In a further embodiment, the fragment is between about one quarter and one half the length of the full-length CTGF protein. "The ability to induce collagen synthesis" must mean the ability to induce extracellular matrix formation via collagen synthesis and differentiation of myo-fibroblasts. CTGF fragments can be obtained either by isolation from natural sources, production by synthetic manufacturing, recombinant genetic engineering techniques, or other techniques available in the field. As used herein, the term "N-terminal" refers to the nucleic acid sequence comprising at least part of the domains of exon 2 and exon 3, beginning at about the full length of the CTGF molecule, and the encoded amino acid sequence, as identified in Figures 3A and 3B. The term "exon 2" refers to the nucleic acid sequence of the N-terminal domain starting approximately the full length of the CTGF molecule, and the corresponding amino acid sequence. The term "exon 3" refers to the nucleic acid sequence of the N-terminal domain of the full-length CTGF molecule, fjp and the encoded polypeptide. As used herein, the term "C-terminal" refers to the nucleic acid sequence comprising at least a portion of the exon 4 and exon 5 domains of the full-length CTGF molecule, and the polypeptide encoded by it, as identified in Figures 3A and 3B. The terms "disorders" and "diseases" as used herein, refer to conditions associated with the expression or activity of CTGF ("diseases or disorders associated with CTGF.) Diseases, disorders, and conditions associated with CTGF. include, but are not limited to, excessive scarring that is the result of acute or repetitive trauma, including surgery or radiation therapy, fibrosis of organs such as kidney, lungs, liver, eyes, heart, and skin, including scleroderma, keloids, and Hypertrophic scarring: Abnormal expression of CTGF has been associated with general tissue scarring, tumor-like growths on the skin, and sustained scarring of blood vessels, leading to impaired ability to carry blood, hypertension, hypertrophy, etcetera. CTGF are different diseases caused by vascular endothelial cell proliferation or migration, such as or cancer, including dermatofibromas, conditions related to abnormal endothelial cell expression, breast carcinoma desmosplasis, angiolipoma, and angioleiomyoma. Other related conditions include atherosclerosis and systematic sclerosis, including atherosclerotic plaques, inflammatory bowel disease, Chrohn's disease, angiogenesis, and other proliferative processes that play central roles in atherosclerosis, arthritis, cancer, and other disease states, involved neovascularization. in glaucoma, inflammation due to illness or injury, including inflammation of the articulacio-lv. of tumors, metastasis of tumor growth, interstitial disease, dermatological diseases, arthritis, including chronic rheumatoid arthritis, arteriosclerosis, diabetes, including diabetic nephropathy, hypertension, and other disorders of the kidney, and fibrosis that is the result of chemotherapy, treatment with radiation, dialysis, and allograft rejection and transplantation. The "fibroproliferative" disorders referred to herein include, but are not limited to, any of the diseases or disorders listed above, eg, kidney fibrosis, scleroderma, pulmonary fibrosis, arthritis, hypertrophic scarring, and atherosclerosis. The fibroproliferative disorders associated with CTGF also include diabetic nephropathy and retinopathy, hypertension, and other disorders of the kidney, disorders associated with angiogenesis, including, but not limited to, blood vessels associated with tumor formation, and other proliferative processes. they play central roles in atherosclerosis, arthritis, and other disease states and, for example, in the skin, cardiac Cr, and pulmonary and renal fibrosis. In general, severe fibrosis involving the kidney, liver, lung, and cardiovascular system is included in the present. There are numerous examples of fibrosis, including the formation of scar tissue after a heart attack, which impairs the heart's ability to pump. Diabetes frequently causes damage / scarring of the kidneys, which leads to a progressive loss of kidney function. Even after surgery, scar tissue can form between internal organs, causing contracture, pain, and, in some cases, infertility. Major organs such as heart, kidneys, liver, lungs, eyes, and skin are prone to chronic scarring, commonly associated with other diseases. Hypertrophic scars (bulky non-malignant tissue) are a common form of fibrosis caused by burns and other traumas. In addition, there are many other fibroproliferative disorders such as scleroderma, keloids, and atherosclerosis, which are respectively associated with general tissue scarring, tumor-like growths on the skin, or sustained scarring of blood vessels, which deteriorates the ability to carry blood. Since CTGF is overexpressed in fibrotic disorders, it represents a very specific target for the development of anti-fibrotic therapeutic products. CTGF can be inhibited through the use of small molecules and neutralizing anti-bodies, for example, in the treatment of fibro-proliferative disorders. It is understood that "fibro-proliferative" refers to any of the above pathological cases, and should not be limited to cell proliferation. "Extra-cellular matrices (ECM)" are multi-component structures, synthesized by, and surrounding different cell types including, for example, endothelial, epithelial, epidermal, and muscular cells. The NDE is formed mostly of collagen and proteoglycans of heparan sulfate. The ECM also contains fibronectin hl, vitronectin, chondroitin sulfate proteoglycans, and smaller proteins. Growth factors are sequestered in these matrices by association with the glycosaminoglycan portion of the heparan sulfate proteoglycans. The "heparin-like" regions of high iduronic acid and high sulfation have been associated with the bFGF binding region of heparan sulfate from human fibroblasts (Turnbull et al., J. Biol. Chem. 267 (15) 10337-10341 , 1992). However, the composition of heparan sulfate in the extra-cellular matrix has not been fully characterized. The phrases "nucleic acid" or "nucleic acid sequence" as used herein, refer to an oligonucleotide, nucleotide, polynucleotide, or fragment of any of these, to DNA or RNA of genomic or synthetic origin, which may be of a single chain or double chain, and may ff represent a sense or anti-sense chain, peptide nucleic acid (PNA), or any material similar to DNA, or similar to RNA, natural or synthetic origin. "Amino acid" or "amino acid sequence" as used herein, refer to a sequence of oligopeptides, peptides, polypeptides, or proteins, or to a fragment, moiety, or subunit of any of these, and to naturally occurring or synthetic molecules. "Hybridization" refers to the process by which a nucleic acid chain is linked to a complementary strand through the formation of base pairs. Hybridization reactions can be sensitive and selective, in such a way that a particular sequence of interest can be identified, even in samples in which it is present at low concentrations. Suitably astringent conditions can be defined by, for example, the salt or formamide concentration in the prior hybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. In particular, astringency can be increased by reducing the salt concentration, increasing the formamide concentration, or raising the hybridization temperature. For example, hybridization under conditions of high stringency may occur in approximately 50 percent formamide, at approximately 37 ° C to 42 ° C. Hybridization can C occur under conditions of reduced stringency in approximately formamide at 35 percent to 25 percent, at approximately 30 ° C to 35 ° C. In particular, hybridization can occur under conditions of high stringency at 42 ° C in 50 percent formamide, 5X SSPE, 0.3 percent SDS, and 200 μg / milliliter of trimmed and denatured salmon sperm DNA. Hybridization can occur under conditions of reduced stringency as described above, but in 35 percent formamide, at a reduced temperature of 35 ° C. the temperature range corresponding to a particular level of astringency can be narrowed by calculating the purine or pyrimidine ratio of the nucleic acid of interest, and adjusting the temperature in accordance therewith. Variations on the above ranges and conditions are well known in the art. The term "substantial amino acid homology" refers to molecules that have a sequence similarity of about 75 percent or more, preferably 85 percent or more, and most preferably 90-95 percent, with a sequence specific. The phrases "percentage of similarity" or "percentage of identity" refer to the percentage of similarity or sequence identity found in a comparison of two or more amino acid or nucleic acid sequences. The percentage of similarity can be determined by methods well known in the art. ^ * The percentage of similarity between the amino acid sequences can be calculated, for example, using the method by 5 clusters. (See, for example, Higgins, D. G. and P. M. Sharp, 1988, Gene, 73: 237-244). Cluster cluster groups are sequenced in clusters by examining the distances between all pairs. Clusters are aligned in pairs, and then in groups. The percentage similarity between two sequences of lv. amino acids, for example, sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of intermediate open space residues in sequence A, minus the number of intermediate open space residues in the sequence B, between the sum of the coincidences of waste between sequence A and sequence B, per hundred. Intermediate open spaces of little or no homology between the two amino acid sequences are not included to determine the percentage of similarity. The percentage of similarity can be calculated by means of other methods known in the art, for example, by means of varying hybridization conditions, and can be calculated electronically using programs such as the MEGALIGN program. (DNASTAR Inc., Madison, Wisconsin). The term "collagen sub-unit" refers to the amino acid sequence of a polypeptide chain of a collagen protein encoded by a single gene, as well as to any derivatives of that sequence, including deletion derivatives, conservative substitutions, etc. ^ A "fusion protein" is a protein in which the peptide sequences of different proteins are covalently linked together. An "anti-sense sequence" is any sequence capable of specifically hybridizing to an objective sequence. The anti-sense sequence can be DNA, RNA, or any mimic or analogous nucleic acid. The term "anti-sense technology" refers to any technology that depends on the specific hybridization of an anti-sense sequence to an objective sequence. The term "functional equivalent", as used herein, refers to a protein or nucleic acid molecule that possesses functional or structural features to the CTGF fragment. A functional equivalent of a CTGF fragment may contain modifications, depending on the need for those modifications for the operation of a specific function. The term "functional equivalent" is intended to include fragments, mutants, hybrids, variants, analogs, or chemical derivatives of a molecule. A molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical fractions that are not normally part of the molecule. These fractions can improve the solubility, absorption, biological half-life, and the like of the molecule. Fractions can alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effects of the molecule, and the like. The fractions capable of mediating these effects are described, for example, in Gennaro, A.R., ed. , 1990, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton PA. In the art, methods for coupling those fractions to a molecule are well known. A "variant", as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The variant may have conservative changes, wherein a substituted amino acid has similar structural or chemical properties, for example, the replacement of leucine with isoleucine. More rarely, a variant may have non-conservative changes, for example, the replacement of a glycine with a tryptophan. Minor analogical variations may also include amino acid deletions or insertions, or both. The guide for determining which amino acid residues can be substituted, inserted, or deleted can be found using computer programs well known in the art, for example, DNASTAR software (DNASTAR Inc., Madison, Wisconsin). Methods for Making Fragments of CTGF Nuclide Acid Sequences Encoding CTGF. In accordance with the invention, nucleotide sequences encoding CTGF or functional equivalents thereof, as described in U.S. Patent No. 5,408,040, can be used to generate recombinant DNA molecules which direct the expression of the full length protein, or a functional equivalent thereof, or alternatively, nucleotide sequences encoding the desired CTGF fragment, for example, in appropriate host cells, a fragment comprising at least a portion of exons 2 or 3 of the CTGF. AlternativelyIt is also possible to use nucleotide sequences which hybridize, under astringent position, to portions of the CTGF sequence, in nucleic acid hybridization assays, Southern and Northern blot analysis, and so on. In yet another method, the DNA molecules encoding CTGF can be isolated by hybridization procedure comprising the classification of "anti-bodies of expression libraries, to detect shared structural features." Due to the inherent degeneracy of the genetic code, other DNA sequences that encode proteins of substantial amino acid homology, or a functionally equivalent amino acid sequence, can be isolated and used in the practice of the invention for the cloning and expression of CTGF or the CTGF fragment. which are capable of hybridizing to the human CTGF sequence under stringent conditions The altered DNA sequences that can be used according to the invention include deletions, additions, or substitutions of different nucleotide residues, giving as a result a sequence encoding the same, or a functionally equitable gene product ivalent.The gene product itself may contain deletions, additions, or substitutions of amino acid residues within the CTGF sequences, which results in a calm change, thereby producing a functionally equivalent protein. These amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the enveloped residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; the positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups, which have similar hydrophilicity values, include the following: leucine, isoleucine, valine; glycine, analin; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine. The DNA sequences of the invention can be designed, for the purpose of altering the sequence of the protein by a variety of ends, including, but not limited to, alterations to modify the processing and expression of the gene product. For example, mutations can be introduced using techniques that are well known in the art, for example, site-directed mutagenesis to, for example, insert new restriction sites. For example, in certain expression systems such as yeast, the host cells f can over-glycosylate the gene product. When such expression systems are used it may be preferable to alter the coding sequence of the CTGF or the CTGF fragment, to eliminate any N-linked glycosylation site. The CTGF sequence or the CTGF fragment can be ligated to a heterologous sequence for encode a fusion protein. For example, to classify peptide libraries it may be useful to encode a chimeric CTGF protein that expresses a heterologous epitope that is recognized by a commercially available antibody. A fusion protein can also be designed to contain a dissociation site located between the CTGF sequence or the CTGF fragment and the heterologous protein sequence (eg, a sequence encoding a PDGF-related growth factor), such that the CTGF or a CTGF fragment can be dissociated away from the heterologous fraction. In the matter those methods are known. The coding sequence of CTGF or a fragment of CTGF can 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, Nucl eic Acids Res. 9 (12): 2807-2817). Alternatively, the protein itself can be produced *? using chemical methods to synthesize the amino acid sequence of CTGF in whole or in part. For example, the peptides can be synthesized by solid phase techniques, dissociated from the resin, and purified by high performance liquid chromatography of preparation. See, for example, Creighton, 1983, Proteins Structures And Molecular Principles, W.H. Freeman and Co., N.Y. pages 50-60. The composition of the synthetic peptides can be formed by analysis or sequencing of amino acids. For example, for the Edman degradation procedure, see, Creighton, 1983, Proteins, Structures and Molecular Principies, W.H. Freeman and Co. , N.Y., pages 34-49. Expression of CTGF or a Fragment of CTGF. For the purpose of expressing a biologically active CTGF fragment, the nucleotide sequence encoding the full-length protein, or a functional equivalent as described above, the CTGF fragment is inserted into an appropriate expression vector, i.e. vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. More specifically, methods that are well known to those skilled in the art may be used to construct expression vectors containing the CTGF sequence or the CTGF fragment, and appropriate transcription / translation control signals. These methods include recombinant DNA techniques in vitro, synthetic techniques and * live recombination / genetic recombination. See, for example, the techniques described by Maniatis et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. A variety of expression vector systems can be used in the host, to express the coding sequence of the CTGF or the CTGF fragment. These include, but are not limited to, microorganisms such as bacteria, transformed with recombinant bacteriophage DNA expression vectors, plasmid DNA or cosmid DNA, which contain the coding sequence of the CTGF or the CTGF fragment; yeast, including Pichia pastoris and Hansenula polymorph, transformed with recombinant yeast expression vectors containing the coding sequence of the CTGF or the CTGF fragment; insect cell systems infected with recombinant virus expression vectors (eg, baculovirus) containing the coding sequence of the CTGF or the CTGF fragment; cell systems of plants infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV), or transformed with recombinant plasmid expression vectors (eg, plasmid) Ti) containing the coding sequence of the CTGF or the CTGF fragment; or animal C cell systems infected with recombinant virus expression vectors (e.g., adenovirus, vaccinia virus, human tumor cells (including HT-1080)), which include cell lines designed to contain multiple copies of CTGF DNA and either stably amplified (CHO / dhfr) or unstable amplified in double minute chromosomes (eg, murine cell lines). As used herein, it is understood that the term "host expression vector systems" and more generally the term "host cells" includes any progeny of the host cell or host expression vector system. It is also understood that although all progeny may not be identical to the stem cell, since mutations can occur during replication, that progeny is 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 used, any of many suitable transcription and translation elements, including constitutive and inducible promoters, can be used in the expression vector. For example, when cloned into bacterial systems, inducible promoters such as pL or the bacteriophage γ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used.; when cloned into insect cell systems, promoters such as the baculovirus polyhedrin promoter can be used; when cloning in plant cell systems, Tm can be used promoters derived from the genome of plant cells (eg, heat shock promoters, the promoter for the small subunit of RUBISCO, the promoter for the binding protein chlorophyll a / b), or plant virus (e.g., the CaMV 35S RNA promoter, the TMV coat protein promoter); when cloned into mammalian cell systems, promoters derived from the genome of mammalian cells (eg, the metallothionein promoter), or from mammalian viruses (eg, the adenovirus late promoter; vaccinia 7.5K); When cell lines containing multiple copies of the CTGF DNA or the CTGF fragment are generated, vectors based on SV40, BPV and EBV can be used with an appropriate selectable marker. In bacterial systems, many expression vectors may conveniently be selected, depending on the intended use for the CTGF or the expressed CTGF fragment. For example, a suitable expression vector in bacteria includes the vector based on T7, as described in Rosenberg, et al., 1987, Gene .56: 125. As a further example, when large quantities of CTGF or CTGF fragment are to be produced, to classify peptide libraries, vectors that direct the expression of high levels of protein products that are rapidly 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), T wherein the coding sequence of the CTGF or the fragment of CTGF can be ligated into the vector in frame with the lac Z coding region, such that a hybrid AS-lac Z protein is produced; vector pIN (Inouye &Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke &Schuster, 1989, J. Biol. Chem. 264: 5503-5509) and the like. The pGEX vectors can also be used to express foreign polypeptides such as CTGF or fragment of CTGF with glutathione S-transferase (GST). In general, these fusion proteins are soluble, and can be easily purified from cells destroyed by the action of the plants, by adsorption to glutathione-agarose globules, followed by leaching in the presence of free glutathione. The pGEX vectors are designed to include thrombin dissociation sites or factor Xa protease, such that the cloned polypeptide of interest can be released from the GST fraction. More generally, where the host is a prokaryote, component cells that are capable of DNA uptake can be prepared from cells harvested after exponential growth, and subsequently treated by the CaC12 method, or alternatively MgCl2 or RbCl , using procedures well known in the art. Where the host cell is a eukaryote, different methods of DNA transfer can be used. These include the transfection of the DNA by means of phosphate calcium precipitates, conventional mechanical procedures, including micro-injection, insertion of a plasmid enclosed in liposomes or the use of virus vectors. Eukaryotic cells can also be co-transformed with DNA sequences encoding the polypeptide of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the simple herpes 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 eukaryotic cells and express the protein. See, Eukaryotic Viral Vectors, 1992, Cold Spring Harbor Laboratory, Gluzman, Ed.). Eukaryotic host cells include yeast, mammalian cells, insect cells and plant cells. In yeast, many vectors containing constitutive or inducible promoters can be used. For a review, see for example, Current Protocols in Molecular Biology, Volume 2, 1988, Ausubel et al., Ed., Greene Publish. Assoc. & Wiley Interscience, Chapter 13; Grant et al., 1987, Methods in Enzymology, Wu and Grossman, Eds., Acad. Press, N.Y., 153: 516-544; Glover, 1986, DNA Cloning, Volume II, IRL Press, Wash., D.C., Chapter 3; Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Berger and Kimmel, Eds., Acad. Press, N.Y., 152: 673-684; and The Molecular Biology of the Yeast Saccharomyces, 1982, Strathern et al., Eds., Cold Spring Harbor Press, Volumes I and II. For example, different launch vectors have been reported for the expression of foreign genes in yeast. Heinemany collaborators, 1989, Nature 340: 205; Rose et al., 1987, Gene 60: 237. In cases where plant expression vectors are used, the expression of the coding sequence of the CTGF or the CTGF fragment can be activated by any of many promoters. For example, viral promoters such as the 35S RNA promoters and the 19S RNA of CaMV (Brisson et al., 1984, Nature 310: 511-514), or the TMV coat protein promoter (Takamatsu et al., 1987) may be used. , EMBO J. 6 ,: 307-311); alternatively, plant promoters such as the small subunit of RUBISCO can be used (Coruzzi et al., 1984, EMBO J. 3: 1671-1680, Broglie et al., 1984, Sci en 224: 838-843); or heat shock promoters can be used, for example, hsp 17.5-E or soy hspl7.3-B (Gurley et al., 1986, Mol Cell. Biol. 6: 559-565). These constructs can be introduced into the plant cells using the Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, micro-injection, electroporation, and so on. For reviews of these techniques, see, for example, Weissbach & amp;; Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pages 421-463; Grierson & Corey, 1988, Plant Molecular Biology, 2nd Edition, ^ Blackie, London, Chapters 7-9. In an insect system, you can use a system of alternative expression to express the CTGF or the fragment of CTGF. In one of these systems, Baculovirus is used as a vector to express foreign genes. The virus then grows in the insect cells. The coding sequence of the CTGF or the CTGF fragment can be cloned into non-essential regions r H (eg, the polyhedrin gene) of the virus, and placed under the control of a Baculovirus promoter. These recombinant viruses are then used to infect insect cells in which the inserted gene is expressed. See, for example, Smith et al., 1983, J. Virol. 46: 584; Smith, patent of the States United No. 4,215,051. In mammalian host cells, many virus-based expression systems can be used. In cases where an adenovirus is used as an expression vector, the coding sequence of the CTGF or CTGF fragment can be ligated to an adenovirus transcription / translation control complex, eg, the late promoter and the leader sequence tripartite This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (for example, the El or E3 region), will result in a recombinant virus that is viable and capable of expressing CTGF or a fragment of CTGF in infected hosts. See, for example,. { Logan & Shenk, 1984, Proc. Nati Acad. Sci. (USA) 81: 3655-3659.

Alternatively, the vaccinia 7.5K promoter can be used. See, for example, Mackett et al., 1982, Proc. Nati Acad. Sci. (USA) 79: 7415-7419; Mackett et al., 1984, J. Virol. 49: 857-864; Panicali et al., 1982, Proc. Nati Acad. Sci. 79: 4927-4931. In another embodiment, the CTGF sequence or the CTGF fragment 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 expression vector pMSXND or the like is used for expression in a variety of mammalian cells, including COS, BHK 293 and CHO cells. Lee and Nathans, 1988, J. Bi ol. Chem. 263: 3521. Specific initiation signals may also be required for the efficient translation of the CTGF coding sequences or the CTGF fragment inserted. These signals include the ATG start 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, additional translational control signals may not be required. However, in cases where only a portion of the CTGF coding sequence is inserted, exogenous translational control signals must be provided, including the initiation codon ff ATG. In addition, the initiation codon must be in phase with the reading frame of the CTGF coding sequence or the CTGF fragment, to ensure translation of the entire insert. These exogenous translational control signals and the initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be improved by the inclusion of appropriate transcription enhancer elements, transcription terminators, and the like. See, for example, Bitter et al., 1987, Methods in Enzymol. 153: 516-544. Additional sequences, ie, leader sequences, etc., can be added to direct the secretion of the CTGF fragments along different secretion trajectories. This can be achieved in many expression systems, using different methods known in the art. In addition, a host cell strain can be chosen that modulates the expression of the inserted sequences, or that modifies and processes the gene product in the specific manner desired. Those modifications (for example, glycosylation) and the processing (eg, dissociation) of the protein products may be important for the function of the protein. Different host cells have specific characteristics and mechanisms for the processing and modification of proteins after translation. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells possessing the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Those mammalian host cells include, but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, HT-1080, and so on. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines stably expressing the CTGF or the CTGF fragment can be designed. Instead of using expression vectors containing viral replication origins, the host cells can be transformed with CTGF DNA or the CTGF fragment controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators). , polyadenylation sites, etc.) and a selectable marker. After the introduction of foreign DNA, designed cells can be allowed to grow for 1-2 days in an enriched medium, and then are switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, and allows the cells to stably integrate the plasmid into their chromosomes, and grow to form foci, which in turn can be cloned and expanded within the cell lines.

Many screening systems can be used, including, but not limited to, thymidine kinase genes from simple herpesvirus (Wigler, et al., 1977, Cell 1.1: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybaiski). , 1962, Proc. Nati, Acad. Sci. (USA) 4.2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22: 817) which can be used in tk, hgprt or aprt cells. , respectively. In addition, anti-metabolite resistance can be used as the basis of selection for the dhfr genes, which confers resistance to methotrexate (Wigler, et al., 1980, Proc. Nati. Acad. Sci. (USA) 77: 3567; Hare, et al., 1981, Proc. Nati. Acad. Sci. (USA) 78.:1527); gpt, which confers resistance to mycophenolic acid (Mulligan &Berg, 1981, Proc. Nati, Acad. Sci. (USA) 78.:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150: 1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30: 147). Recently, additional selectable genes have been described, namely trpB, which allows cells to use indole instead of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman &Mulligan, 1988, Proc. Nati, 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 Communications in Molecular Biology, Cold Spring Harbor Laboratory). f The isolation and purification of polypeptides expressed in host cells of the invention can be by any conventional means such as, for example, chromatographic preparation separations and immunological separations, such as those involving the use of monoclonal or polyclonal antibodies. Identification of Transf ectants or Transformants that Express the CTGF or a Fragment of CTGF. Host cells that contain the coding sequence, and that express the biologically active gene product, can be identified by means of 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 transcripts of CTGF mRNA or of the CTGF fragment in the host cell; and (d) detecting the gene product as measured by means of an assay, or by means of its biological activity. In the first approach, the presence of the CTGF coding sequence or the CTGF fragment inserted into the expression vector can be detected by DNA-DNA or DNA-RNA hybridization, using probes comprising nucleotide sequences that are homologous to the coding sequence of CTGF or of the CTGF fragment, respectively, or portions or derivatives thereof. In the second approach, the recombinant expression host / host system can be identified and selected based on the presence or absence of certain "marker" gene functions (eg, antibiotic resistance, methotrexate resistance). , transformation phenotype, body formation of baculovirus occlusion, etc.). For example, in a preferred embodiment, the CTGF coding sequence is inserted into a sequence of vector neomycin resistance marker genes, and recombinants containing 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 another promoter that is used to control the expression of the CTGF coding sequence. The expression of the marker in response to induction or selection indicates the. expression of the coding sequence of CTGF. In the third approach, the transcriptional activity for the CTGF coding region or the CTGF fragment can be evaluated by hybridization assays. For example, RNA can be isolated and analyzed by Northern blotting, using a probe homologous to the CTGF coding sequence or the CTGF fragment, or particular portions thereof. Alternatively, total nucleic acids can be extracted from the host cell, and can be tested for hybridization to those probes, ff The fourth approach involves detection of the CTGF gene product or biologically active, or immunologically reactive CTGF fragment. Many assays can be used to detect the activity of CTGF, including, but not limited to, those assays described in U.S. Patent No. 5,408,040. Dissociation of CTGF Protein from Longi Complete to Produce the Fragment of CTGF. After expression of the full-length CTGF protein, the protein can be dissociated by any number of proteolytic enzymes known to one of ordinary skill in the art, to result in the CTGF fragments of the present invention. For example, the cysteine-free bridge between the N-terminal and C-terminal moieties of CTGF is susceptible to chymotrypsin, using methods available in the art. Methods for Modulating and Inhibiting the Activity of CTGF Fragments As described above, the CTGF fragments described in this invention are a critical determinant of extracellular matrix deposition. The present invention provides methods for the prevention and treatment of complications associated with CTGF, by regulating, modulating, and / or inhibiting the activity and / or expression of such fragments, or if desirable, increasing activity of such fragments. More specifically, the methods of the present invention provide for the administration of a therapeutically effective amount of an agent that regulates, modulates, and / or inhibits the extra-cellular matrix producing activity of the N-terminal fragments of the CTGF, as desired. , to treat, prevent or ameliorate diseases or disorders associated with the expression and activity of CTGF. Anti-bodies. In one embodiment of the present invention, a method involves the administration of a therapeutically effective amount of an anti-body that specifically reacts with the CTGF fragments of the present invention. Anti-bodies specifically reactive with CTGF are described in U.S. Patent No. 5,783,187 and PCT publication, WO 96/38172. CTGF anti-bodies can be generated using methods well known in the art. Such anti-bodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain antibodies, as well as Fab fragments, including F (ab ') 2 and Fv fragments. The fragments can be produced, for example, by means of a Fab expression library. Especially preferred are neutralizing anti-bodies, ie, those that inhibit the formation of dimers, for therapeutic use. A target polypeptide, such as CTGF or an agent that modulates the activity and / or expression of CTGF, can be evaluated to determine regions of high immunogenicity. The methods of analysis and the selection of epitopes are well known in the art. See, for example, Ausubel, et al., 1988, Current Protocols in Molecular Biolosv. The analysis and selection can also be carried out, for example, by means of different software packages, such as the LASERGENE NAVIGATOR software. (DNASTAR; Madison, Wisconsin). The peptides or fragments that are used to induce the anti-bodies must be antigens, but they are not necessarily biologically active. Preferably, a fragment or peptide antigen has at least 5 amino acids in length, more preferably, at least 10 amino acids in length, and most preferably, at least 15 amino acids in length. It is preferable that the fragment or peptide which induces the anti-body is identical to at least a portion of the amino acid sequence of the target polypeptide, for example, CTGF. A peptide or fragment that resembles at least a portion of the naturally occurring target polypeptide sequence can also be fused to another protein, for example, orifice limpet hemocyanin (KLH), and anti-bodies against the molecule can be produced chimerical Methods for the production of anti-bodies are well known in the art. For example, different hosts, including goats, rabbits, rats, mice, humans, and others, can be immunized by injection with the target polypeptide or any immunogenic fragment or peptide thereof.

Depending on the species of the host, different adjuvants can be used to increase the immune response. These adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among the adjuvants that are used in humans, BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum are especially preferable. Monoclonal and polyclonal anti-bodies can be prepared using any material that allows the production of anti-body molecules by continuous cell lines in culture. Techniques for in vivo and in vi tro production are well known in the art. See, for example, Pound, J.D., 1998, Immuno Chemical Protocols, Human Press, Totowa NJ; Harlow, E. and D. Lane, 1988, Antibodies, A Laboratorv Manual, Cold Spring Harbor Laboratory, New York. The production of chimeric anti-bodies is also well known, as is the production of single-chain anti-bodies. See, for example, Morrison, S.L. et al., 1984, Proc. Nati Acad. Sci. 81: 6851-6855; Neuberger, M.S. et al., 1984, Nature 312: 604-608; Takeda, S. et al., 1985 Nature 314: 452-454. Antibodies with related specificity, but of different idiotypic composition, can be generated, for example, by intermixing the chains of random combination immunoglobulin libraries. See, for example, Burton D.R., 1991, Proc. Nati Acad. Sci. 88: 11120-11123. Antibodies can also be produced by inducing in vivo production in the lymphocyte population, 5 or by classifying immunoglobulin libraries or panels of highly specific binding reagents. See, for example, Orlandi, R. et al., 1989, Proc. Nati Acad. Sci. 86: 3833-3837; Winter, G. and C. Milstein, 1991, Nature 349: 293-299). Fragments of anti-C bodies containing specific binding sites for the target polypeptide can also be generated. These fragments of anti-bodies include, but are not limited to, fragments of F (ab ') 2 / that can be produced by digestion of pepsin from the anti-body molecule, and Fab fragments, which can be generated by 5 reduction of the disulphide bridges of the F (ab ') fragments 2- Alternately, Fab expression libraries can be constructed, to allow the rapid and easy identification of the monoclonal Fab fragments with the desired specificity. See, for example, Huse, W.D., et al., 1989 Science 0 254: 1275-1281. Anti-bodies can be tested for their anti-target polypeptide activity, using a variety of methods well known in the art. Different techniques can be used to classify to identify anti-bodies that have the desired specificity, including different immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), including direct ELISAs and capture by ligands, radio - immunoassays (RIAs), immunoblotting, and selection of fluorescent activated cells (FACS). Many protocols for competitive binding or immuno-radiometric assays are well known in the art, using either polyclonal or monoclonal antibodies with established specificities. See, for example, Harlow and Lane. These immunoassays typically involve the measurement of complex formation between the target polypeptide and a specific anti-body. A two-site immunoassay, based on monoclonal antibodies, using monoclonal antibodies reactive to two non-interfering epitopes in the target polypeptide is preferred, but other assays, such as the competitive binding assay, may also be employed. See, for example, Maddox, D.E., et al., 1983, J Exp Med 158: 1211. The present invention contemplates the use of anti-bodies specifically reactive with a CTGF polypeptide or fragments thereof., which neutralize the biological activity of the CTGF fragments of the present invention. The anti-body administered in the method can be the anti-body or intact antigen that fixes the fragments thereof, such as Fab, F (ab ') 2 and Fv fragments, which are capable of fixing the epitope determinant. The anti-bodies used in the method can be polyclonal or, more preferably, monoclonal anti-bodies. Monoclonal anti-bodies with different epitopic specificities are made from antigen containing fragments of the protein, by methods well known in the art. ^ See, for example, Kohler et al., Na ture 256: 494; Ausubel et al., Supra. In the present invention, therapeutic applications include those using "human" or "humanized" antibodies directed to CTGF or fragments thereof. Humanized anti-bodies are anti-bodies, or anti-body fragments, which have the same binding specificity as an anti-body stem (ie, typically of mouse origin), and increased human characteristics. Humanized anti-bodies can be obtained, for example, by intermixing the chains, or by the use of phage display technology. For example, a polypeptide comprising a heavy or light chain variable domain of a non-human anti-body specific for a CTGF, is combined with a repertoire of chain variable domains (light or heavy) complementary to human. Hybrid pairs specific for the antigen of interest are selected. The human chains of the selected pairs can then be combined with a repertoire of human complementary (heavy or light) domains and the humanized anti-body polypeptide dimers can be selected for binding specificity for an antigen. The techniques described for the generation of humanized anti-bodies, which may be used in the method of the present invention, are described in, for example, U.S. Patent Nos. 5,565,332; 5,585,089; 5,694,761; and 5,693,762. In addition, the techniques described for the production of human anti-bodies in transgenic mice are described in, for example, U.S. Patent Nos. 5,545,806 and 5,569,825. Anti-sense oligonucleotides. The present invention provides a therapeutic approach that is directly interconnected with the translation of CTGF messages, and specifically messages of full length CTGF (wherein the full length protein is then dissociated, to form a CTGF fragment of the present invention) or the CTGF fragment (collectively "CTGF mRNA"), in the protein. More specifically, the present invention provides a method wherein anti-sense nucleic acids or ribozymes are used to bind to, or dissociate CTGF mRNA. Anti-sense RNA or DNA molecules bind specifically with the RNA message of the targeted gene, disrupting the expression of the protein product of that gene. The anti-sense is fixed to the messenger RNA, forming a double-stranded molecule that can not be transferred through the cell. In addition, chemically reactive groups, such as iron-linked ethylenediaminetetraacetic acid (EDTA-Fe), can be attached to an anti-sense oligonucleotide, causing dissociation of the RNA at the hybridization site. These and other uses of anti-sense methods to inhibit the translation of genes are well known in the art. See, for example, Marcu-Sakura, 1988, Anal. Biochem 177: 278. More specifically, in one embodiment of the invention, the sequence of an anti-sense polynucleotide useful for inhibiting the expression of CTGF mRNA can be obtained by comparing orthologous gene sequences (sequences that are conserved between species). , or transcripts of orthologous genes, and identifying the highly conserved regions within those sequences.The similarity in nucleic acid sequences can be determined by methods and algorithms that are well known in the art.These procedures and algorithms include, for example , a BLAST program (Basic Local Alignment Research Tool in the National Biological Information Center), ALIGN, AMAS (Multiple Sequence Analysis Aligned), and AMPS (Alignment of Multiple Protein Sequences) .When selecting the preferred length for a given polynucleotide, different factors must be considered to achieve the characteristics most favorable icas. In one aspect, the polynucleotides of the present invention are at least 15 base pairs (bp) in length, and preferably from about 15 to about 100 bp in length. More preferably, the polynucleotides are from about 15 to about 80 bp in length, and even more preferably, the polynucleotides of the present invention are from about 15 to about 60 bp in length. Shorter polynucleotides, such as 10 to below 15 mers, although they offer higher cell penetration, have gene specificity plus f. low. In contrast, polynucleotides longer than 20 to about 30 bp offer better specificity, and show decreased uptake kinetics within cells. See, Stein et al., "Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression," Cohen, ed., McMillan Press, London (1988). The accessibility to the RNA target sequences of the transcript is also of importance for the regions of curve formation and the orthologous sequences in the target ARs, thus offering promising objectives. In this description, the term "polynucleotide" encompasses both oligomeric and nucleic acid fractions of the naturally occurring type, such as the deoxyribonucleotide and ribonucleotide structures of AD? and AR ?, and man-made analogs that are capable of binding to the nucleic acid found in nature. The polynucleotides of the present invention can be based on the ribonucleotide and deoxyribonucleotide monomers linked by phosphodiester linkages, or by means of analogs linked by methyl phosphonarose, phosphorothionate and other linkages. These may also comprise monomer fractions having altered base structures or other modifications, but still retaining the ability to bind to RA structures? of naturally occurring transcripts. Such polynucleotides can be prepared by methods well known in the art, for example, by the use of commercially available machines and reagents such as those available from Perkin-Elmer / Applied Biosystems (Foster City, California). For example, polynucleotides specific to a directed transcript are synthesized in accordance with standard methodology. DNA polynucleotides modified by phosphorothionate are typically synthesized in automated DNA synthesized in automated DNA synthesizers available from a variety of manufacturers. These instruments are capable of synthesizing amounts of nano-moles of polynucleotides as large as 100 nucleotides. The shorter polynucleotides synthesized by modern instruments are often suitable for use without further purification. If necessary, the polynucleotides can be purified by polyacrylamide gel electrophoresis, or reverse phase chromatography. See, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Volume 2, Chapter II, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Phosphodiester-linked polynucleotides are particularly susceptible to the action of nucleases in serum or internal cells, and therefore, in a preferred embodiment, the polynucleotides of the present invention are analogues linked to phosphothionate or methyl phosphonate, which have been shown to be resistant to the nuclease. Those of ordinary skill in the art can easily select other links for use in this invention. These modifications can also be designed to improve cellular uptake and stability of the polynucleotides. A suitable carrier for the administration of a polynucleotide may include, for example, vectors, antibodies, pharmacological compositions, binding or targeting proteins, or viral delivery systems for enriching the sequence within the target cell or tissue. A polynucleotide of the present invention can be coupled to, for example, a binding protein that recognizes endothelial cells or tumor cells. After the administration, a polynucleotide of the present invention can be directed to a recipient cell or tissue, such that improved expression of, for example, cytokines, transcription factors, G-protein coupled receptors, tumor suppressor proteins, and apoptosis initiation proteins. The delivery of anti-sense molecules and the like can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. The different viral vectors that can be used for gene therapy, as taught herein, include adenovirus, herpes virus, vaccinia, or preferably an RNA virus such as a retrovirus. Many known retroviruses can transfer or incorporate a gene for a selectable marker, such that transduced cells can be identified and generated. By inserting a sequence of polynucleotides of interest into the viral vector, together with another gene encoding the ligand for a receptor in a specific target cell, for example, the vector is specific to the target.Retroviral vectors can be made specific to the target by means of inserting, for example, a polynucleotide that encodes a sugar, a glycolipid or a protein.The preferred targeting is done by the use of an anti-body to direct the retroviral vector.Those of experience in the art will know of, or can quickly find out without undue experimentation, specific polynucleotide sequences that can be inserted into the retroviral genome to allow specific delivery to the target of the retroviral vector containing the anti-sense polynucleotide Since the recombinant retroviruses are defective, they require assistance with the purpose of producing particles of vect This assistance can be provided, for example, by the use of helper cell lines containing plasmids that encode all the structural genes of the retrovirus, under the control of regulatory sequences within LTR. These plasmids are missing a nucleotide sequence that enables the packaging mechanism to recognize an RNA transcript for encapsidation. Helper cell lines that have deletions of the packaging signal include, but are not limited to, 2, PA317, and PA12. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into cells in which the packaging signal is intact, for the structural genes are replaced by other genes of interest, the vector can be packaged and the vector virion can be produced. Alternatively, NIH 3T3 or other tissue culture cells can be transfected directly with the plasmids encoding retroviral structural genes gap, pol, and env, by means of conventional calcium phosphate transfection. These cells are then transfected with the plasmid of the vector containing the genes of interest. The resulting cells release the retroviral vector into the culture medium. Another targeted delivery system for antisense molecules is a colloidal dispersion system. Colloidal dispersion systems include complexes of macromolecules, nano-capsules, microspheres, globules and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles that are useful as delivery systems in vivo and in vitro. It has been shown that large unilamellar vesicles (LUV), which vary in size from 0.2-4.0 um can encapsulate a substantial percentage of an aqueous pH regulator that contains large macromolecules. The RNA, DNA and intact virions can be encapsulated inside the aqueous interior, and sent to the cells in a biologically active form. In order for a liposome to be an effective gene transfer vehicle, the following characteristics must be present: (1) encapsulation of the genes of interest at high efficiency, while not compromising its biological activity; (2) preferential and substantial attachment to a target cell compared to non-target cells; (3) sending the aqueous content of the vesicle to the cytoplasm of the target cell at high efficiency; and (4) exact and effective expression of genetic information. Inhibitors of Small Molecules. The present invention also provides a method in which small molecules that inhibit the activity of the CTGF fragment of the present invention are identified and used. The identification of small molecules that inhibit the activity of the CTGF fragment can be driven by different classification techniques. For the classification of the compounds, the assay will provide a detectable signal associated with the binding of the compound to a protein or cellular target. Depending on the nature of the assay, the detectable signal may be absorbance or light emission, plate formation, or other convenient signal. The result can be qualitative or quantitative. For the classification of the compounds to see the specific binding, different immunoassays can be used to detect the human anti-bodies (or primate) fixed to the T cells. In this way, one can use anti-hlg tagging, for example, anti-hlgM, hlgG or combinations thereof, to detect the specifically fixed anti-human body of the galactosyl epitope. Different labels can be used such as radioisotopes, enzymes, fluorescers, chemoluminiscers, particles, and so on. There are numerous commercially available cases that provide anti-hlg labeling, which can be employed in accordance with the manufacturer's protocol. Different protocols can be used to classify a library of chemical compounds. To some degree, the selection of the appropriate protocol will depend on the nature of the preparation of the compounds. For example, the compounds can be attached to individual particles, nails, membranes or the like, wherein each of the compounds can be segregated. In addition, the amount of compound available will vary, depending on the method used to create the library. In addition, depending on the nature of the binding of the compound to the support, one may be able to release aliquots of a compound, for the purpose of conducting a series of tests. In addition, the manner in which the compounds are tested will be affected by the ability to identify the compound shown to have activity. Where the compounds are individually on a surface in a grid, such that at each grid site one knows what the composition is, one can provide a cell plot that is organized in a similar manner to a grid, and can be place in register with the compounds fixed to the solid surface. Once the plot and the solid substrate are in register, one can release the compounds from the surface, in accordance with the manner in which the compounds are bound. After sufficient time for the compounds to bind to the proteins on the cell surface, one can wash the cell plot to remove non-specifically bound compounds. One or more washes may be involved, where the washes may provide different degrees of astringency, depending on the desired degree of affinity. After the washings are finished, then the blood or the mammalian plasma can be added, and incubated for sufficient time for cyto-toxicity. The plasma or blood can then be removed, and the plates can be observed, where the nature of the compound can be determined by virtue of the position of the grid. Of course, the plasma or blood should be free of any components that would naturally remove the cells from the plot. Since the preparation process can be repeated, one can prepare a plurality of solid substrates, where the same compounds are prepared at comparable sites, so that classification with the same or different cells can be repeated, to determine the activity of the individual compounds. In some cases, the identity of the compound can be determined by a nucleic acid label, using the polymerase chain reaction for the amplification of the label. See, for example, WO 93/20242. In this case, compounds that are active can be identified by taking the lysate, and introducing the lysate into a polymerase chain reaction medium comprising specific primers for the nucleic acid tag. After expansion, one can sequence the nucleic acid tag, or determine its sequence by other means, which will indicate the synthetic procedure that was used to prepare the compound. Alternatively, one can have labeled particles where the labels can be released from the particle, and provide a binary code describing the synthetic procedure for the compounds fixed to the particle. See, for example, Ohlmeyer, et al., PNAS USA (1993) 90: 10922. These labels can conveniently be a homologous series of alkylene compounds, which can be detected by electron capture by gas chromatography. Depending on the nature of the linking group, one can provide partial release of the particles, such that the particles can be used 2 or 3 times, before identifying the particular compound. Although for the most part the libraries have been described, any large group of compounds can be classified analogously, as long as the CTGF epitope can bind to each of the compounds. In this way, compounds from different sources, both natural and synthetic, including macrolides, oligopeptides, ribonucleic acids, dendrimers, etc., can also be classified in an analogous manner. The formation of a plaque in the assay demonstrates that the attachment of the library member to the cell, usually a surface protein, does not interfere with the binding of the CTGF epitope to an anti-body, that the immune complex is sufficiently stable to initiate the complement cascade, and that the member has a high affinity for the target. In PCT Publication WO 9813353 other classification methods are described for obtaining small molecules that modulate the activity of the CTGF fragments of the present invention. Pharmaceutical Formulations and Administration Routes In order to identify small molecules and other agents useful in the present methods to treat or prevent a renal disorder by modulating the activity and expression of CTGF, CTGF and biologically active fragments can be used of it to classify the therapeutic compounds, in any of a variety of classification techniques. The fragments that are used in these sorting tests can be free in solution, fixed to a solid support, originating in a cell surface, or located intra-cellularly. The blocking or reduction of biological activity, or the formation of binding complexes between the CTGF and the agent being tested, can be measured by methods available in the art. In the matter other techniques for the classification of drugs are known, which provide a high throughput classification of compounds that have adequate binding affinity to CTGF, or to other target polypeptides useful for modulating, regulating, or inhibiting the expression and / or activity of CTGF. For example, micro-configurations carrying test compounds can be prepared, used, and analyzed, using methods available in the art. See, for example, Shalon, D. et al., 1995, PCT Application No. WO 95/35505, Baldeschweiler et al., 1995, PCT Application No. WO 95/251116.; Brennan, T.M. et al., 1995, U.S. Patent No. 5,474,796; Heller, M.J. et al., 1997, U.S. Patent No. 5,605,662. The identification of small molecules that modulate the activity of CTGF can also be conducted by means of other different classification techniques, which can also serve to identify anti-bodies1 and other components that interact with CTGF, and that can be used as drugs and therapeutic products in the present methods. See, for example, Enna, ^^ S.J. and collaborators, eds. , 1998, Current Protocols in Pharmacovs, John Wiley and Sons. The assays will typically provide detectable signals, associated with the binding of the compound to a cellular protein or target. The fixation can be detected by, for example, fluorophores, enzyme conjugates, and other detectable labels well known in the art. See, for example, Enna et al. The results can be qualitative or quantitative. To classify the compounds by specific binding, numerous immunoassays can be used to detect, for example, anti-human or primate bodies attached to the cells. In this manner, one can use anti-hlg tagging, for example, anti-hlgG, hlgG or combinations thereof, to detect anti-human body specifically fixed from the galactosyl epitope. Different labels can be used such as radioisotopes, enzymes, fluorescers, chemoluminiscers, particles, and so on. There are numerous commercially available cases that provide anti-hlg labeling, which can be employed in accordance with the manufacturer's protocol. For the classification of the compounds to see their cyto-toxic effects, a wide variety of protocols can be used to ensure that one has the desired activity. One will normally use cells, which may be naturally occurring or modified cell lines, or the like. The cells can be prokaryotic or eukaryotic. For example, if one is interested in a pathogen, where it does not matter to which epitope the conjugate of the compound is fixed, one can combine the pathogenic cells with each of the compounds in the presence of an anti-cyto-toxic system dependent on the -body, to determine the cyto-toxic effect. One can perform this assay either before or subsequent to the determination of the effect of the different candidate compounds on the cells of the host to whom the compound would be administered. In this way, one would obtain a differential analysis between the affinity for the pathogenic objective, and the affinity for the host cells that could be found, based on the mode of administration. In some situations, one would be interested in a particular cell state, such as an activated state, as it might be present with T cells in autoimmune diseases, transplantation, and the like. In this situation one would first classify the compounds to determine those that bind to the quiescent cell, and to those compounds that are not binding to the quiescent cells, and classify the remaining candidate compounds for cyto-toxicity to the activated cells. One could then classify the other cells present in the host that the compounds could find, to determine their cyto-toxicity effect. Alternatively, one could use cancer cells and normal cells to determine if any of the compounds have greater affinity for cancer cells, compared to normal cells. Again, one can classify the library of compounds to see the binding to normal cells and determine the effect. Those compounds that are not cyto-toxic to normal cells can then be classified to see their cyto-toxic effect on cancer cells. Even where there is some cyto-toxicity for normal cells, in the case of cancer cells, where there is sufficient differentiation in cyto-toxic activity, one may be willing to tolerate the lowest cyto-toxicity for normal cells, where otherwise the compound is effective with cancer cells. Instead of using cells that are naturally obtained, one can use cells that have been modified by recombinant techniques. In this way, one can employ cells that can grow in culture, which can be modified by up-regulating or down-regulating a particular gene. In this way, one would have cells that differ in terms of a single protein on the surface. One can then differentially assay the library, as to the effect of the library members on the cells for which the particular protein is present or absent. In this way, one can determine whether the compound has specific affinity for a particular surface membrane protein, as distinct from any of the proteins present in the surface membrane. One can differentiate between cells by using g of anti-bodies that bind to a particular surface membrane protein, where the anti-bodies do not initiate the cyto-toxic effect dependent on complement, for example, using different species, isotypes or combinations thereof. By adding the anti-bodies, blocking antisera or monoclonal anti-bodies, to a portion of the cells, those cells will not have the target protein available for attachment to the library member. In this way one creates comparative cells that differ in their response, based on the inability in a single protein group. Although antibodies will usually be the most convenient reagent to use, other specific binding entities, which provide the same function, may be employed. To be used in the assay to determine binding, one can use a cyto-toxic antibody-dependent system. One can use synthetic mixtures of the ingredients, where only those components necessary for the cyto-toxic effect are present. This may be desirable where the components of blood or plasma may adversely affect the test results. In addition, although a cell plot is an extremely convenient way to classify large numbers of candidates, they may also find use of other techniques. These The techniques include the use of multiple well plates, and the different devices that are used for the preparation of the combination gene pool, such as bald, tea bags, and so on. One could grow the cells separately in relation to the nature of the different devices, wherein the device can then contact the cells, or cause the cells to grow in the device. The device can be immersed in an appropriate culture, seeded with the cells, or otherwise provided for contact between the cells and the candidate compound. After the addition of the cyto-toxic agent, one could then analyze the destruction by the action of the plants, in a variety of ways. For example, FACS can be used to distinguish between living and dead cells, the release of sup 51 Cr, or the detection of an intracellular compound in the supernatant, can be used to detect active compounds. In addition, one may wish to know whether the compound has agonist or antagonist activity. The present assay techniques provide a rapid way to determine those compounds present in the library, which bind to the target protein. Once one has substantially narrowed the number of candidate compounds, one can use more sophisticated assays to detect the activity of the compound itself. In this way, one can perform a rapid classification to determine affinity and specificity of fixation, followed by a more intensive classification to determine the activity. There are different techniques to determine the activity, where the t cells can be modified, in such a way that a marker gene will be activated that will provide a detectable signal. Conveniently, the signal may be associated with the production of a dye, the production of a surface membrane protein, which can be detected with labeled anti-bodies, or the secretion of a protein that can be detected in the supernatant, by any of a variety of techniques. For example, the gene that is expressed may be modified luciferase to have a leader sequence, for the purpose of being secreted, whereby it can then be classified for the formation of light generation by the use of an appropriate substrate. Different protocols can be used to classify the library. To a certain degree, this will depend on the nature of the preparation of the compounds. For example, the compounds can be fixed individual particles, nails, membranes, or the like, wherein each of the compounds can be segregated. In addition, the amount of compound available will vary, depending on the method used to create the library. In addition, depending on the nature of the binding of the compound to the support, one may be able to release aliquots of a compound, for the purpose of conducting a series of tests. In addition, the manner in which the compounds are tested will be affected by the ability to identify the compound shown to have activity. Where the compounds are individually on a surface in a grid, such that at each grid site one knows what the composition is, one can provide a cell plot that is organized in a similar manner to a grid, and can be place in register with the compounds fixed to the solid surface. Once the plot and the solid substrate are in register, one can release the compounds from the surface, in accordance with the manner in which the compounds are bound. After sufficient time for the compounds to bind to the proteins on the cell surface, one can wash the cell plot to remove non-specifically bound compounds. One or more washes may be involved, where washes can provide different degrees of astringency, depending on the degree of affinity desired. After the washings have been completed, then the blood or mammalian plasma can be added, and incubated for sufficient time for cyto-toxicity. The plasma or blood can then be removed, and the plates can be observed, where the nature of the compound can be determined by virtue of the position of the grid. Of course, the plasma or blood should be free of any components that would naturally remove the cells from the plot. Since the preparation process can be repeated, one can prepare a plurality of solid substrates, where the same compounds are prepared at comparable sites, so that classification with the same or different cells can be repeated, to determine the activity of the individual compounds. In some cases, the identity of the compound can be determined by a nucleic acid label, using the polymerase chain reaction for the amplification of the label. See, for example, PCT application No. WO 93/20242. In this case, the compounds that are active can be determined by taking the lysate, and introducing the lysate into a polymerase chain reaction medium comprising specific primers for the nucleic acid label. Then, from the expansion, one can sequence the nucleic acid tag, or determine its sequence by other means, which will direct the selection of the method that is used to prepare the compound. Alternatively, one can have labeled particles, where the labels can be released from the particle, and provide a binary code describing the synthetic procedure for the compounds fixed to the particle. See, for example, Ohl eyer, et al., 1993, PNAS USA 90: 10922. These labels can conveniently be a homologous series of alkylene compounds, which can be detected by electron capture by gas chromatography. Depending on the nature of the linker group, one can provide partial release of the particles, such that the particles can be used 2 or 3 times, before identifying the particular compound. Although for the most part the libraries have been described, any large group of compounds can be classified analogously, as long as the CTGF epitope can bind to each of the compounds. In this way, compounds from different sources, both natural and synthetic, including macrolides, oligopeptides, ribonucleic acids, dendrimers, etc., can also be classified in an analogous manner. The formation of a plaque in the assay demonstrates that the attachment of the library member to the cell, usually a surface protein, does not interfere with the binding of the CTGF epitope to an anti-body, that the immune complex is sufficiently stable to initiate the complement cascade, and that the member has a high affinity for the target. The present methodology can be used in any situation where one has a cell target to be eliminated, particularly those cell targets that have little or no CTGF epitope. In this way, the cell target can be a prokaryote, which is pathogenic. Different organisms include, for example, mycobacteria, Yersinia, Pseudomonas, Bordetella pertussis, Treponema pallidum, Neisseria gonorrhea, Streptococcus, Hemophilus infl uenza, and so on. Other pathogens include eukaryotes, particularly fungi, such as Candida, Histoplasma, et cetera, and protozoa, for example, ffj Giardia. In addition, viruses that provide surface membrane proteins in infected cells can also be targeted by the compounds present, where the cells that are classified have become vitally infected. Host cells can also serve as targets, wherein the cells are either abnormal or act in an adverse manner to the host or host treatments. For example, cancerous tissues that can be distinguished from normal tissue can serve as a target for the compounds present. T or B cells associated with autoimmune diseases or associated with GVHD or transplant rejection may also serve as targets. Aberrant cells can also serve as targets, regardless of their nature, as long as they can be distinguished from normal cells. In this way, lesions due to psoriasis, lymphoma cells, bacterial, fungal, parasitic cells, infected by viruses, may be targets of the present products. In addition, the present compounds can find application where one wishes to remove a portion of cells, without the removal of all cells, such as cells expressing a differentiation marker, such as subsets of T cells, activated platelets, endothelial cells, cells that express hormone or cytokine receptors.

Other classification methods for obtaining small molecules that modulate CTGF activity can be found, for example, in PCT Application No. WO 98/13353. Compounds / Molecules. The present invention provides methods for treating and preventing disorders, diseases and disorders associated with CTGF, by modulating, regulating, or inhibiting the activity of CTGF or CTGF fragments of the present invention. These methods may comprise administering a therapeutically effective amount of a compound that blocks the binding interactions, or which blocks the enzymes involved in the CTGF signal transduction path. More specifically, the present invention provides a method for inhibiting CTGF activity by administering compounds that block the induction of CTGF. Compounds that modulate the expression of the CTGF gene and / or the activity of CTGF in the method of the invention, include agents that cause an elevation in the cyclic nucleotides in the cell. Other compounds that can block the induction of CTGF can be identified, in accordance with the methods of the present invention, using the classification methods described above. In one embodiment, the invention provides a method for identifying a compound or agent that modulates the activity, for example, production of extracellular matrix proteins, induction of myo-fibroblast differentiation, induction of collagen synthesis, a fragment of CTGF, for example a fragment encoded by exons 2 and 3, as set forth in Figure 3. The method includes contacting an agent of interest, such as a peptide, small molecule, polypeptide, peptide, with cells (e.g., fibroblasts) and a fragment of CTGF known to have the desired activity, e.g., production of collagen, and measure the ability of cells to produce collagen by any means known to one skilled in the art ( see the EXAMPLES). The ability of the cells to produce collagen is then compared to the ability of a population of adequate cell control to produce collagen in the absence of the agent or compound. The term "agent" or "compound", as used herein, describes any molecule, for example, a protein, polypeptide, or pharmaceutical product, with the ability to affect the growth of a cell. The agent can be anything that is known or suspected to be capable of affecting the growth of the cells. The agent includes peptide fragments of the CTGF polypeptide. The agents include synthetic chemical agents, biochemical agents, cells, extracts, homogenates and conditioned medium. The test agent can also be a combination library to classify a plurality of compounds. The compounds identified in the method of the invention can also be evaluated, detected, cloned, sequenced, and the like, either in solution, or after fixation to a solid support, by any method usually applied to the detection of a specific DNA sequence, such as PCR, oligomer restriction (Saiki et al., Bio / Technology 3: 1008-1012, 1985), allele-specific oligonucleotide probe analysis (ASO) (Conner et al., Proc. Nati. Acad. Sci. USA 80: 278, 1983), oligonucleotide ligation assays (OLAs) (Landegren et al., Science 241: 1077, 1988), and the like. Molecular techniques for DNA analysis have been reviewed (Landegren et al., Science 242: 229-237, 1988). The candidate agents cover numerous chemical classes. These can be organic molecules, preferably small organic compounds having a molecular weight of more than 50, and less than about 2,500 Daltones. The candidate agents comprise the functional groups necessary for structural interaction with proteins, particularly hydrogen fixation, and typically include at least one amino, carbonyl, hydroxyl, or carboxyl group, preferably at least two functional chemical groups. Candidate agents frequently comprise cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures, substituted with one or more of the above functional groups. Candidate agents are also among the biomolecules, including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogues or combinations thereof. The candidate agents may be polypeptides, or polypeptides produced by site-directed or random mutagenesis of a synthetic or naturally occurring nucleic acid sequence. In yet another embodiment of the present invention, the method provides for the administration of molecules that interrupt the subsequent translational modification of full-length CTGF, or block the activation of an inactive precursor of CTGF. As described herein, exposure of mensangio cells to TGF-β resulted in the marked appearance of additional bands at 28-30 kDa, which correspond in size to the carboxy terminal and terminal amino moieties of the molecule. Full-length CTGF. As described above, treatment with TGF-β can result in the production of proteases or other factors capable of dissociating the full-length molecule. Molecules that inhibit CTGF activity can be identified using the classification methods provided herein. Pharmaceutical Formulations and Administration Routes Administration Routes. Compositions comprising CTGF modulators, ie, anti-bodies, anti-sense oligonucleotides, small molecules, and other compounds as described herein, can be administered to a human patient per se, or in pharmaceutical compositions that understand, where appropriate, suitable carriers or ff excipients. The present invention contemplates methods of treatment in which agents that modulate or regulate the expression or activity of CTGF or fragments thereof, are administered to a patient in need, in amounts suitable to treat or prevent the activity or expression of the fragment of CTGF. The present methods of treatment and prevention may comprise administering an effective amount of the agent to a subject that is preferably a mammalian subject, and most preferably, a human subject. In a preferred embodiment, the administered mammalian subject and agent are of homologous origin. More preferably, the subject and the agent administered are of human origin. An effective amount can be quickly determined by routine experiments, as well as the most effective and convenient route of administration and the most appropriate formulation can be determined. In the matter, different formulations and drug delivery systems are available. See, for example, Gennaro, A.R., ed. , 1990, Reminston 's Pharmaceuti cals Sciences, 18th edition, Mack Publishing Co. , Easton PA. Suitable routes of administration may include, for example, oral, rectal, transmucosal, or intestinal administration and parenteral application, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intravenous injections. nasal, or intra-ocular. The composition can be administered in a local, rather than systematic, manner. The pharmaceutical compositions of the present invention can be manufactured by any of the methods well known in the art, such as by conventional mixing, dissolving, granulating, dragee-making, leaching, emulsifying, encapsulating, entrapping, or lyophilizing processes. As noted above, the compositions of the present invention may include one or more physiologically acceptable carriers such as excipients and auxiliaries, which facilitate processing of the active molecules into preparations for pharmaceutical use. The appropriate formulation depends on the route of administration. For injection, for example, the composition can be formulated in aqueous solutions, preferably in physiologically compatible pH regulators, such as Hank's solution, Ringer's solution, or physiological saline pH regulator. For transucosal administration, appropriate penetrators are used in the formulation for the barrier to infiltrate. These penetrators are generally known in the art. For oral administration, the compounds can be rapidly formulated by combining the active compounds with pharmaceutically acceptable carriers, well known in the art. These carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject. The compounds can also be formulated in rectal compositions such as suppositories, or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides. Pharmaceutical preparations for oral use can be obtained as solid excipients, optionally by grinding a resulting mixture, and processing the granule mixture, after the addition of 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, corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate. The dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions, which optionally may contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures may be used. . Dyestuffs or pigments can be added to tablets or dragee coatings, for identification or to characterize different dose combinations of the active compounds. Pharmaceutical preparations for oral administration include push-mold capsules made of gelatin, as well as sealed, soft capsules, made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-mold capsules may contain the active ingredients in combination with the 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 can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in doses suitable for that administration. For administration by inhalation, the compounds for use in accordance with the present invention are conveniently applied in the form of an aerosol spray presentation of pressurized packages or nebulizers, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane , carbon dioxide, or any other suitable gas. In the case of a pressurized aerosol, the appropriate dose unit can be determined by means of providing a valve for sending a measured quantity. Capsules and cartridges of, for example, gelatin can be formulated for use in an inhaler or insufflator. These typically contain a mixture of powders of the compound and a suitable powder base, such as lactose or starch. Compositions formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion, may be presented in unit dosage form, for example, in ampules or in multi-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 formulating agents such as suspending, stabilizing and / or dispersing agents. Formulations for parenteral administration include aqueous solutions of agents that effect CTGF activity or fragments thereof, in water soluble form. Suspensions of the active compounds can be prepared as appropriately oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, and synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain stabilizers or suitable agents that increase the solubility of the compounds, to allow the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form, for constitution with a suitable vehicle, for example, sterile, pyrogen-free water, before use. The compositions of the present invention can also be formulated as a depot preparation. These long-acting formulations can be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as barely soluble derivatives, for example, as a soluble. Pharmaceutical carriers for the hydrophobic molecules of the invention can include co-solvent systems comprising, for example, yl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system can be the VPD co-solvent system. VPD is a 3 percent weight / volume, 8 percent weight / volume yl alcohol solution of non-polar surfactant polysorbate 80, and 65 percent weight / volume polyethylene glycol 300, conformed to volume in absolute ethanol. The VPD co-solvent system (VPD: 5W) consists of VPD diluted 1: 1 with a 5 percent dextrose solution in water. This co-solvent system is effective in dissolving hydrophobic compounds, and produces low toxicity after routine administration. Naturally, the proportions of a cosolvent system can vary considerably, without destroying its solubility and toxicity characteristics. In addition, the identity of the co-solvent components can be varied. For example, other non-polar, low toxicity surfactants can be used instead of the polysorbate 80, the fraction size of the polyethylene glycol can be varied, other biocompatible polymers can replace the polyethylene glycol, for example, polyvinylpyrrolidone, and other sugars or Polysaccharides can substitute for dextrose. Alternatively, other application systems for hydrophobic molecules may be employed. Liposomes and emulsions are well known examples of carriers or carriers for hydrophobic drugs. It is also possible to use certain organic solvents such as dimethisulfoxide, although usually at the cost of greater toxicity. Additionally, the compounds can be applied using sustained release systems, such as semi-permeable matrices of solid hydrophobic polymers containing the effective amount of the composition to be administered. Different materials of sustained release are established and available for those with experience in the field. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks to more than 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed. Effective dose. For any composition that is used in the present methods of treatment, initially a therapeutically effective dose can be estimated, using a variety of techniques well known in the art. For example, in a cell culture assay, a dose can be formulated in animal models, to achieve a concentration range in circulation that includes the IC50, as determined in the cell culture. Where inhibition of CTGF activity is desired, for example, the concentration of the test compound which achieves maximum mean inhibition of CTGF activity can be determined. Appropriate dose ranges for human subjects can be determined using data obtained from cell culture assays and other animal studies. A therapeutically effective dose refers to that amount of the molecule that results in relief of symptoms, or an extension of survival in a subject. The toxicity and therapeutic efficacy of these molecules can be determined by standard pharmaceutical procedures in cell cultures, or in experimental animals, for example, by determining the LD50 (the lethal dose for 50 percent of the population), and the ED50 (the effective therapeutic dose in 50 percent of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the ratio of LD50 / ED50. Molecules that exhibit high therapeutic indices are preferred. Doses preferably fall within a range of circulating concentrations that include ED50 with little or no toxicity. Doses may vary within this range, depending on the dosage form used, and the route of administration used. The exact formulation, the route of administration and the dose will be chosen in view of the specificities of the condition of a subject. The amount of dose and the range can be adjusted individually to provide plasma levels of the active fraction, which are sufficient to modulate or regulate the activity of the CTGF as desired, ie, the minimum effective concentration (MEC). The MEC will vary for each compound, but can be estimated from, for example, in vi tro data, such as the concentration needed to achieve 50-90 percent activity of CTGF, to induce bone growth using the assays. described in the present. The doses needed to achieve MEC will depend on the individual characteristics and the route of administration.

The compositions should be administered using a regimen that maintains plasma levels on the ECM by approximately 10-90 percent of the duration of the treatment, preferably approximately 30-90 percent of the duration of the treatment, and more preferably approximately 50-90 percent. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to the plasma concentration. The amount of composition administered, of course, will be dependent on many factors, including, but not limited to, the weight of the particular subject, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician. Packaging The compositions may, if desired, be presented in a pack or dosing device, which may contain one or more dosage unit forms containing the active ingredient. The package may, for example, comprise sheet metal or plastic, such as a bubble pack. The package or dosing device may be accompanied by instructions for administration. Compositions comprising a compound of the invention, formulated in a compatible pharmaceutical carrier, placed in an appropriate container, and labeled for the treatment of an indicated condition can also be prepared. The suitable conditions indicated on the label could include the treatment of disorders or diseases in which the induction of cartilage or bone is desired, the healing of lesions, neuro-protection, fibrosis of > Cf kidney, diabetes or similar. EXAMPLES The following examples are provided only to illustrate the claimed invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended to be illustrations of the individual aspects of the invention only, and methods that are functionally equivalent are within the scope of the invention. In fact, various modifications of the invention will be apparent, in addition to those described herein, for those skilled in the art, by the following description and accompanying drawings. It is intended that these modifications fall within the scope of the appended claims. Example 1: CTGF Fragments Stimulate Extra-Cell Matrix Synthesis To prepare fragments of CTGF, human recombinant CTGF (full length) was directed by chymotrypsin to give a CTGF fragment. Fragments of recombinant CTGF were also produced by expressing either or both exons 2 and 3 of CTGF. A continuous line of cultured normal rat kidney fibroblasts (NRK), designated as clone NRK-49F, was obtained from the American Type Culture Collection (ATCC) to produce cell cultures. Skin fibroblasts were established from the human forehead from explanted cultures. The ^, cell cultures were maintained in modified medium of Dulbecco (DME) containing 2.5% fetal bovine serum and 2.5% Nu-serum I (Collaborative Biomedical Products, Bedford, Massachusetts, United States) and passed before the confluence. To examine fibroblast collagen synthesis, mono-layers of arrested growth of NRK and human skin skin fibroblasts were prepared by seeding 10,000 cells / well in 48-well plates and allowing cells to grow to confluence in 5 to 7 days in DME and 2.5% fetal bovine serum / Nu-serum. Mono-layers of fibroblasts were seeded with serum in DME containing 25 mM HEPES and ITS premix (Collaborative Biomedical) for 1 to 8 days. Ascorbic acid (50 mg / ml) and biological agents (CTGF fragments) were then added. Collagen synthesis was determined by measuring the incorporation of 3H-proline into extracellular / cell-associated, salt-precipitated collagen, resistant to pepsin, using a quantitative assay for the 24 hour end of the 48-hour treatment. As indicated in figure 2, CTGF fragments comprising exons 2 and 3 of collagen synthesis stimulated by CTGF in NRK fibroblasts with 1 ng / ml of TGF-b. In contrast, carboxyl-terminal CTGF did not stimulate collagen synthesis. Example 2: Fragments of CTGF Induce Differentiation of Myo- Fibroblasts Fragments of CTGF and cell cultures were prepared as described above. To examine the induction of myo-fibroblasts by the CTGF fragments, NRK detached growth mono-layers for frontal skin fibroblasts were prepared and treated as described above. After the treatments, monocaps in 48 cavities plate were washed twice with TBS and fixed in methanol at -20 'C for 10 minutes before being processed for immuno-histological detection of alpha-smooth muscle. Immuno-histological detection was conducted. After fixation, the monolayers of cells were washed twice with TBS and blocked for 30 minutes with 10% horse serum / 2% milk in TBS. The cells were then incubated for one hour with monoclonal mouse alpha-smooth muscle anti-mouse actin IgG (Clone 1A4, Sigma Chemical) to a solution of 1: 200 in horse / milk / TBS serum. After three washes with TBS, then mono-cell layers were incubated for one hour with bio-tin-lysed horse anti-mouse IgG (Vector Labs, Burlingame, California, United States) to a solution of 1: 200 in serum of horse / milk / TBS. The monolayers of cells were then washed and incubated for 30 minutes with a strepvidin-biotin complex conjugated with alkaline phosphate (Dako, Glostrup, Denmark). After washing, the alkaline phosphatase was visualized with a fast red substrate (Vector Red, Vector Labs) in the presence of 1 mM levamisole. Mono-layers of processed cells were then examined under a microscope in relation to myelin-fibroblasts positive for alpha-smooth muscle actin marked in red and the number of myo-fibroblasts was counted per cavity and the selected microscopic fields were photographed. As indicated in FIG. 1, the results of the assay of collagen synthesis of fibroblasts are mimicked by the assay of myoblast induction. Specifically, the CTGF fragments of the present invention were able to induce differentiation of myoblasts, compared to terminal carboxyl CTGF fragments, which were unable to induce such differentiation. Example 3: Neutralizing Anti-Bodies Anti-CTGF Blocks DNA Synthesis, Collagen Synthesis and Induction of TGF-β-induced Myo-Fibroblasts Specific anti-CTGF anti bodies were formed against recombinant, biologically active human CTGF , produced in a baculovirus expression system using methods known in the art. The anti-bodies were prepared in goats and tested with respect to the neutralization of CTGF activity directly or on DNA or collagen synthesis induced by TGF in NRK fibroblasts. The goat anti-bodies exhibited activity in the assays to neutralize the action of TGF-β. In these assays, goat anti-CTGF anti-bodies were able to block DNA synthesis. In addition, as demonstrated in Figure 5, the CTGF anti-bodies were able to block collagen synthesis and the formation of myo- ^ 0 fibroblasts induced by TGF-β. It was observed that the amount of anti-body required to block collagen synthesis was considerably less than the amount needed to block DNA synthesis. Western blot assay and competition ELISA assays both indicated that the majority of the antibodies in this preparation were directed against the domain N-terminal of CTGF. This suggested that the two domains may be responsible for stimulating different biological activities. Example 4: Anti-CTGF Anti-Bodies Specific to the N-Terminal Domain of CTGF Selectively Block Collagen Synthesis Anti-CTGF domain specific antibodies were prepared by affinity chromatography using N-terminal or C-terminal CTGF domains . These domains were prepared from intact CTGF by chymotrypsin-limited digestion. The domains were separated from one another by affinity chromatography on heparin sepharose. The N-terminal domain is not bound to heparin, while the C-terminal domain of CTGF contains heparin binding activity and is retained on the heparin sepharose. These domains were pure, having less than 0.1% contamination with intact CTGF, based on Western spotting analysis. The individual domains were then coupled to Affigel 10 at a concentration of approximately 0.5 mg / ml gel. Total anti-CTGF IgG (goat) was then absorbed into the affinity resin, and the specifically bound anti-bodies were eluted. These anti-bodies were tested in f ^ Western blots to determine the specificity of their reactivity. IgG's reactive with only the N-terminal domain or only the C-terminal domain of CTGF were isolated from the total pond using techniques known in the art. The anti-bodies were then tested in neutralization assays using CTGF. The results of these studies indicated that anti-bodies directed against the N-terminal domain of CTGF selectively inhibited collagen synthesis, but not DNA synthesis, as demonstrated in Figure 6. In contrast, targeted anti-bodies against the C domain of CTGF they selectively inhibited DNA synthesis, but not collagen synthesis. The data indicated that different regions of the CTGF molecule may be responsible for signaling different biological activities. To confirm and extend these results, the biological activities of the isolated domains with intact CTGF and with TGF-β were compared, as noted below. Example 5: The N-Terminal Domain of CTGF Stimulates the Production of Extra-Cell Matrix N-terminal and C-terminal domains of CTGF were prepared using techniques known in the art. First, as described above, pure N-terminal and C-terminal domains were prepared by proteolytic digestion of intact, biologically active CTGF using chymotrypsin, since almost exclusively N-terminal and C-terminal domains were produced, without fragmentation. - (smaller formats.) A second method for generating pure C-terminal and N-terminal domains involved expressing only limited regions of the CTGF reading frame, which encoded only the C-terminal domain or only the N-terminal domain. was achieved by PCR amplification of portions of the open reading frame, and introducing either a stop codon in the cysteine-free region to produce only the N-terminal domain or by cloning the portion of the open reading frame that encodes only the domain C-terminal, starting in the AYRLED sequence in the cysteine-free region to the GP67 baculovirus shuttle vector.This produced a chimeric protein containing a signal peptide from the GP67 virus gene that directed the synthesis of the desired recombinant protein (or fragment) to the endoplasmic reticulum, thereby ensuring secretion. After purification, the isolated domains generated by the various methods were compared in a bio-assay with NRK fibroblasts. The results of these studies confirmed the previous observations with anti-CTGF anti-bodies domain-specific. The N-terminal domains produced either by proteolytic digestion of intact CTGF or by direct recombinant expression were fully active as inducers of collagen synthesis and induction of myo-fibroblasts, as indicated in figures 7 and 8. Conversely, the C-terminal domains produced by any of the methods were also fully active in the DNA synthesis assay. The data demonstrated that the individual CTGF domains retained full biological activity, and can act independently of each other to stimulate specific biological effects on target cells. At optimal concentrations, the individual domains induced a biological response comparable to intact CTGF or TGF-β. This strongly indicated that the mitogenic (DNA synthesis) and matrigenic (extracellular matrix synthesis, such as collagen synthesis) activities of TGF-β are mediated via CTGF and their respective domains. Other growth factors can be used with the CTGF fragments of the present invention to increase the inductive activity of CTGF on extracellular matrix production. For example, the peptide growth factor, IGF-2, was evaluated for its effect on the inductive activity of the collagen and myo-fibroblast phenotype of CTGF. Concentrations of 2 ng / ml or greater of IGF-2 in the presence of CTGF resulted in a large increase in collagen synthesis and the myo-fibroblast phenotype, as shown in Figure 9. Various modifications and variations of the methods and described systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to specific embodiments, it should be understood that the invention, as claimed, should not be unduly limited to such specific embodiments. In fact, various modifications of the modes described to carry out the invention that are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims. All references cited herein are incorporated herein by reference in their entirety.

Claims (17)

1. CLAIMS 1. A polypeptide fragment of connective tissue growth factor (CTGF) having the ability to induce extracellular matrix synthesis, collagen synthesis, and / or myo-fibroblast differentiation.
2. 2. A fragment of claim 1, comprising an amino acid sequence encoded by at least exon 2, as indicated in figure 3. 3. A fragment of claim 1, comprising an amino acid sequence encoded by at least exon 3, as indicated in figure
3. 3.
4. 4. A fragment of claim 1, comprising an amino acid sequence encoded by at least exons 2 and 3, as indicated in figure 3.
5. 5. A polynucleotide that encodes a fragment as in claim 1.
6. 6. An anti-body that specifically binds to a CTGF fragment of claim 1.
7. 7. An anti-sense molecule that is linked to a nucleic acid sequence that encodes a fragment of CTGF of claim 1.
8. 8. A method for treating a disease or disorder associated with CTGF, comprising administering to a subject having or at risk of having a disease or disorder associated with CTGF, an anti-body of the reivin 6
9. 9. The method of claim 8, wherein the disease or disorder is a fibroproliferative disorder / disease. The method of claim 8, wherein the disease or disorder is selected from the group consisting of kidney fibrosis, scleroderma, pulmonary fibrosis, hepatic fibrosis, arthritis, hypertrophic scarring, atherosclerosis, diabetic nephropathy and retinopathy, hypertension, kidney, disorders related to angiogenesis, fibrotic disorders of the skin, and cardiovascular disorders. 11. A method for treating a disorder or disorder associated with CTGF, which comprises administering to a subject having or at risk of having a disease or disorder associated with CTGF, an anti-sense molecule of claim 7. 12. A method of identifying an agent or a compound that modulates the activity of an N-terminal fragment of CTGF, comprising: contacting a myo-fibroblast cell with a test agent and with an N-terminal CTGF fragment under conditions that allow the components to interact; and compare the ability of the cell to differentiate in the presence of the agent with the ability of a cell to differentiate in the absence of the agent, where a difference in the differentiating capacity of the cells is indicative of an agent or a compound that modulates the activity of differentiation of a fragment of CTGF. < ^ 13. The method of claim 12, wherein the modulation is activity inhibition. The method of claim 12, wherein the modulation is activity stimulation. 15. A method of identifying an agent or a compound that modulates the activity of an N-terminal CTGF fragment, comprising: contacting a cell with a test agent and with an N-terminal CTGF fragment, under conditions that allow the components to interact; and comparing the ability of the compound or agent to modulate the production of extra-cellular matrix in the presence of the agent with the ability of an agent or a compound to modulate extra-cellular matrix production in the absence of the agent, where a difference in production of extra-cellular matrix is indicative of an agent or a compound that modulates the activity of a CTGF fragment. 16. The method of claim 12, wherein the modulation is activity inhibition. 17. The method of claim 12, wherein the modulation is activity stimulation.