EP1991276A2 - Treatment of tendinopathy by inhibition of molecules that contribute to cartilage formation - Google Patents

Abstract

The invention provides methods for treating tendinopathy. The disorders treated or prevented include, for example tendinitis, tendonitis, tendinosis, paratendinitis, tenocynovitis, tendon overuse injury and trauma, peritendinitis, paratenonitis, or other tendon degenerative disorders. The disclosed therapeutic methods include administering to a patient an inhibitor of molecules involved in cartilage or fibrocartilage formation in tendinopathic tendon in an amount effective to treat or prevent a tendon degenerative disorder, slow tendon deterioration, restore tendon healthy structure, stimulate tendon regeneration, and/or maintain tendon mass and/or quality.

Description

TREATMENT OF TENDINOPATHY BY INHIBITION OF MOLECULES THAT CONTRIBUTE TO CARTILAGE FORMATION

DESCRIPTION OF THE INVENTION PRIOR APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 60/779,165, filed March 3, 2006, the contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The technical field of the invention relates to the treatment of tendinopathy by reducing the production of cartilage and/or fibrocartilage tissue in damaged tendons. The invention further relates to the inhibition of molecules involved in the production of cartilage and/or fibrocartilage in damaged tendons.

BACKGROUND OF THE INVENTION

[0003] Tendinopathy is a general term used to describe various types of tendon disorders. The term "tendinitis" — which means "inflammation of the tendon" — is often used to describe tendon problems, but inflammation is rarely the cause of tendon pain. Most commonly, tendon pain is actually a symptom of a series of microtears in the connective tissue in or around the tendon, more properly called tendinosis. Other tendon disorders include tendon pain due to collagen degeneration with fiber disorientation, increased mucoid ground substance in tendon, calcification, tendon overuse, vascularization, aging, and rubbing of tendon against a body protuberance. Tendinopathy is used by a growing number of tendon experts to describe these conditions, often characterized as tendinitis, tendinosis, paratendinitis, tenosynovitis, paratenonitis, tendon overuse injuries, and trauma, collectively. Khan et al., Sports Med 27:393-408 (1999).

[0004] Normal tendon tissue is composed of densely packed connective tissue with regularly arranged bundles of collagen fibers running in the same direction in primary, secondary, and tertiary fiber bundles that have high tensile strength. Interspersed among these fibers are the flat, tapered tenocytes that synthesize the viscous extracellular matrix (ECM) rich in type I collagen. The type I collagen bundles provide the tendon's flexibility and structural support. In healthy tendons, a dynamic equilibrium exists between synthesis and degradation of the ECM. Tendinopathy, however, adversely affects this equilibrium and results in structural rearrangement and disorganization of the collagen fibers. The disorganization of the collagen bundles gives the tendon the appearance of cartilage. Fibroblasts, myofibroblasts, neovascularization, and pathological accumulation of glycosaminoglycans (GAGs) in the ECM are clearly noticeable in tendinopathic tissue. Tallon et al., Med Sci Sports Exerc 33(12): 1983-90 (2001); Khan et al., Sports Med 27(6) :393-408 (1999). Metabolic and morphological changes in a damaged tendon result in pain and susceptibility to tears and rupture.

[0005] Currently, the degradation and remodeling of a damaged tendon's ECM is believed to be caused by the release of metalloproteinases from resident connective tissue cells and invading inflammatory cells that are capable of degrading matrix macromolecules such as aggrecan, decorin, and biglycan. These metalloproteinases include MMPs (Matrix Metalloproteinases), ADAMs (A Disintegrin And Metalloproteinase), and ADAMTs (A Disintegrin And Metalloproteinase with Thrombospondin Motifs, such as aggrecanase). Riley G., Expert Rev MoI Med 7 (5)-Λ -23 (2005); Rees et al., Biochem J 350: 180-188 (2000). However, clinical trails of broad-spectrum inhibitors of MMPs were unsuccessful in treating osteoarthritis, which has pathologic similarities to tendinopathy. Clark et al., Expert Opin Ther Targets 7(1):19-34 (2003). Therefore, it is not likely that the inhibition of metalloproteinases will be effective in treating tendinopathy. Indeed, in highly stressed tendons such as the supraspinatus and achilles tendons, a minimum level of metalloproteinase activity might be needed to provide the tendon with an optimal level of ECM. Riley G., Expert Rev MoI Mec/7(5):1-23 (2005).

[0006] Presently, various standard non-operative and operative treatments of tendinopathy exist. The non-operative measures include rest, cryotherapy, activity modification, physiotherapy, nonsteroidal anti-inflammatory drugs (NSAIDs), and corticosteroids. Rest and activity modification may help patients with some of these conditions, but there remains a significant clinical population who are not treatable with these therapies. Despite widespread use, oral anti-inflammatory medications have not proven to be useful in controlled studies, and can have undesireable side effects. Some studies further suggest that non-steroidal medication may actually have an adverse effect on the healing process because, by alleviating pain, they allow the patient to ignore early symptoms of tendinopathy.

[0007] Corticosteroids are normally used to reduce inflammation in tissues, but the use of such drugs for treating tendinopathy is not recommended, particularly because corticosteroids inhibit collagen synthesis. Several studies have observed a short-term improvement in patients treated with cortisone, but studies that followed patients beyond one year revealed a high symptom recurrence rate and an equivocal efficacy rate. Cortisone injections also carry the risk of tendon rupture, infection, skin depigmentation, and subdermal atrophy. In diabetic patients, the injection may cause hyperglycemia.

[0008] Surgical measures for tendon repair include debridement and repair of the damaged tendons. However, open or arthroscopic surgery has many potential complications such as deep infection, damage to neurovascular structures, and scar formation. The surgery is also expensive and carries the additional risks associated with regional or general anesthesia.

[0009] Therefore, there is still a need for treatment protocols for tendon related injuries superior to existing treatments. Injuries or other damage to flexible tendon tissues are slow to heal, and can take months or even years to completely repair. Tendon related injuries have a significant impact on society. Overuse injuries account for nearly 7 percent of all injury-related physician office visits in the United States. Woodwell et al., Adv Data 346:1-44 (2004). Based on the information from the U.S. Department of Labor, Bureau of Labor Statistics, such injuries amount to significant loss of work time (see http://www.bls.gov/lif).

SUMMARY OF THE INVENTION

[0010] This invention provides a novel approach to the treatment of tendinopathy. By performing transcriptional profiling of overused rat tendons, it has now been discovered that the expression levels of cartilage-specific genes such as aggrecan, versican, Col2a1 (type Il collagen), and Sox9 are increased in injured tendons. These results suggest that the injured tendon tissue is converted into fibrocartilage as a result of the overuse. Unlike tendon, which is composed primarily of type I collagen, fibrocartilage contains type Il collagen and large proteoglycans, such as aggrecan and versican. The formation of cartilage and fibrocartilage within a tendon is detrimental to tendon repair because the cartilage tissue disrupts the closely packed type I collagen fibers of tendon. This disruption reduces the tendon's tensile strength and elasticity.

[0011] Accordingly, the invention provides methods of treating tendinopathy in a patient by reducing the formation of cartilage-specific proteoglycans in the patient's tendon tissue. The invention also provides uses of compounds that reduce the formation of cartilage-specific proteoglycans for the manufacture of medicaments for treating tendinopathy.

[0012] In some embodiments, the methods and uses include reducing the activity of an enzyme involved in the synthesis of aggrecan and/or versican in the patient's tendon tissue. In particular embodiments, the methods and uses include reducing the activity of chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CS-GalNAcT-1) and/or galactosamine polypeptide N- acetylgalactosaminyltransferase 1 (GalNT-1) in the patient's tendon tissue. In other embodiments, the methods and uses include reducing the activity of one or more other enzymes involved in cartilage proteoglycan synthesis, including, but not limited to, UDP-D-xylose:core protein β-D-xylosyltransferase, GaI transferases, GIcA transferases, GIcNAc transferases, sulfotransferases, chondroitin sulfate synthases, and heparan sulfate sulfotransferases.

[0013] The activity of enzymes and other proteins involved in the production of cartilage structural proteins may be reduced by directly or indirectly inhibiting the function of the enzyme or other protein, or may be reduced by inhibiting the expression of the enzymes or other proteins themselves. [0014] The invention also provides methods of treating tendinopathy in a patient by reducing the expression of cartilage-specific structural proteins in the patient's tendon tissue. The invention further provides uses of compounds that reduce the expression of cartilage-specific structural proteins for manufacture of a medicament for treating tendinopathy. In some embodiments, the expression of cartilage specific structural proteins, such as, but not limited to, aggrecan, syndecan 3 (syn-3), versican, and type Il collagen, is directly inhibited. In other embodiments, the activity of transcription factors involved in the expression of cartilage-specific genes is reduced. These transcription factors and signal transduction proteins include, for example, Sox9.

[0015] In other embodiments, the invention includes methods of identifying a compound for treating tendinopathy comprising administering a test compound to a subject in need of treatment and measuring the ability of the agent to inhibit the activity of an enzyme involved in the synthesis of glycoglycosaminoglycans (GAGs) in tendon tissue.

[0016] In certain embodiments, methods of identifying a compound for treating tendinopathy or assessing treatment comprise: (1) providing at least one sample component selected from the group consisting of CS-GalNAcT-1 , CS- GalNAcT-2, heparan sulfate (glucosamine) 3-O-sulfotransferase 1 (Hs3st1), GaINT- 1 , and Sox9; (2) combining the sample with a test compound; (3) measuring the activity of the sample component in response to the test compound ; and (4) determining whether the test compound of inhibits the activity of the sample component. [0017] Additional objectives and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the following detailed description and appended claims.

[0018] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0019] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In Figures 1-7, SST stands for supraspinatus tendon; PT stands for patellar tendon non-injured internal control; R stands for animals subjected to overuse protocol (Soslowsky et al., J. Shoulder Elbow Surg. 9:79-84 (2000)); C stands for control animals not subjected to overuse protocol; time course is 1 , 2, and 4 weeks; and BM stands for bone marrow.

[0021] Figure 1 shows gene expression profiles for Col2a1 (type Il collagen), in overused tendons (Figure 1A) over four weeks using Affymetrix RAE230 2.0 gene chips. The expression levels of the Col2a1 gene were also measured in normal musculoskeletal tissues as indicated (Figure 1 B). The expression profiles indicate that Col2a1 is highly expressed both in cartilage tissue when compared to normal tendon tissue and in overused tendon tissue when compared to normal tendon tissue.

[0022] Figure 2 shows gene expression profiles for Agc1 (aggrecan), in overused tendons (Figure 2A) over four weeks using Affymetrix RAE2302.0 gene chips. The expression levels of the Agc1 gene were also measured in normal musculoskeletal tissues as indicated (Figure 2B). The expression profiles indicate that Agc1 is highly expressed both in cartilage tissue when compared to normal tendon tissue and in overused tendon tissue when compared to normal tendon tissue.

[0023] Figure 3 shows gene expression profiles for Sox9 (sry-type high mobility group box 9), in overused tendons (Figure 3A) over four weeks using Affymetrix RAE2302.0 gene chips. The expression levels of the Sox9 gene were also measured in normal musculoskeletal tissues as indicated (Figure 3B). The expression profiles indicate that Sox9 is highly expressed both in cartilage tissue when compared to normal tendon tissue and in overused tendon tissue when compared to normal tendon tissue.

[0024] Figure 4 shows gene expression profiles for Cspg2 (versican), in overused tendons (Figure 4A) over four weeks using Affymetrix RAE230 2.0 gene chips. The expression levels of the versican gene were also measured in normal musculoskeletal tissues as indicated (Figure 4B). The expression profiles indicate that versican is highly expressed both in cartilage tissue when compared to normal tendon tissue and in overused tendon tissue when compared to normal tendon tissue. [0025] Figure 5 shows gene expression profiles for chondroitin sulfate N-acetylgalactosaminyltransferase (CS-G al N AcT- 1) in overused tendons (Figure 5A) over four weeks using Affymetrix RAE230 2.0 gene chips. The expression levels of the CS-GalNAcT-1 gene were also measured in normal musculoskeletal tissues as indicated (Figure 5B). The expression profiles indicate that CS- GalNAcT-1 is highly expressed both in cartilage tissue when compared to normal tendon tissue and in overused tendon tissue when compared to normal tendon tissue.

[0026] Figure 6 shows gene expression profiles for GalNT-1 (GaINTI-UDP- N-acetyl-alpha-D-galactosamine:polypeptide-N-acetylgalactosaminyltransferase 1) in overused tendons (Figure 6A) over four weeks using Affymetrix RAE230 2.0 gene chips. The expression levels of the GalNT-1 gene were also measured in normal musculoskeletal tissues as indicated (Figure 6B). The expression profiles indicate that GalNT-1 is highly expressed in overused tendon tissue when compared to normal tendon tissue.

[0027] Figure 7 shows gene expression profiles for Hs3st1 (heparan sulfate (glucosamine) 3-O-sulfotransferase 1) in overused tendons (Figure 7A) over four weeks using Affymetrix RAE230 2.0 gene chips. The expression levels of the Hs3st1 gene were also measured in normal musculoskeletal tissues as indicated (Figure 7B). The expression profiles indicate that Hs3st1 is highly expressed in overused tendon when compared to normal tendon and in cartilage tissue when compated to normal tendon tissue. BRIEF DESCRIPTION OF THE SEQUENCES

[0028] The nucleotide and amino acid sequences of CS-GalNAcT-1 are available in Genbank under the following accession numbers: human (NM_018371); mouse (N M_172753); and rat (XIVL224757).

[0029] The nucleotide and amino acid sequences of CS-GalNAcT-2 are available in Genbank under the following accession numbers: human (NM_018590); mouse (NM_030165); and rat (XM_232316).

[0030] The nucleotide and amino acid sequences of GalNT-1 are available in Genbank under the following accession numbers: human (NM_020474); mouse (NM_013814); and rat (NM_124373).

[0031] The nucleotide and amino acid sequences of aggrecan are available in Genbank under the following accession numbers: human (NM_001135); mouse (NM_007427); and rat (NM_022190).

[0032] The nucleotide and amino acid sequences of Col2a1 are available in Genbank under the following accession numbers: human (NM_001844); mouse (NM_031163) and rat (NM_012929).

[0033] The nucleotide and amino acid sequences of Sox9 are available in Genbank under the following accession numbers: human (NM_000346); mouse (NM_011448); and rat (XM_343981).

[0034] The nucleotide and amino acid sequences of versican are available in Genbank under the following accession numbers: human (NM_004385); mouse (XM_488510) and rat (XM_215451).

[0035] The nucleotide and amino acid sequences of heparan sulfate (glucosamine) 3-0-sulfotransferase 1 are available in Genbank under the following accession numbers: human (NM_005114), mouse (NML.010474), and rat (NM_053391).

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention provides methods for treating tendinopathy by reducing the activity of one or more molecules involved in cartilage and/or fibrocartilage formation in tendon tissues.

[0037] For the present invention to be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description of the invention.

[0038] As used herein, the term "tendinopathy" includes all pathologies that arise in and around tendons, including, but not limited to, tendinitis, tendonitis, tendinosis, paratendinitis, tenosynovitis, tendon overuse injury and trauma, perintendinitis, and paratenonitis.

[0039] As used herein, the term "tendon defect" refers to any tendon disorder including, but not limited to, tendinopathy.

[0040] As used herein, the term "damaged tendon" refers to a tendon tissue suffering from tendinopathy or a tendon defect.

[0041] As used herein, the term "overused" tendon refers to a tendon suffering from a tendon defect or tendinopathy as a result of overuse, as opposed to direct trauma.

[0042] As used herein, the term "patient" or "subject" refers to any person or animal who is susceptible to, suffers from, or is in the process of recovering from, a tendon defect and is in need of treatment. [0043] As used herein, the term "tendinitis" refers to tendonitis (an alternative spelling), tendinopathy, or inflammation of the tendon.

[0044] As used herein, the term "tendinosis" refers to tendinopathy or any degenerative condition where microtears occur in tendon that weaken a tendon, often causing pain, stiffness, and loss of strength.

[0045] As used herein, the term "tendon injury" refers to tendinopathy or any condition in which the tendon tissue becomes defective or degenerative.

[0046] As used herein, the term "small molecule" includes any chemical or other moiety, other than polypeptides and nucleic acids, that can act to affect biological processes.

[0047] As used herein, the terms "treating" or "treatment" refer any administration or application of remedies for disease in a mammal, including a human, and includes inhibiting the disease. It includes arresting disease development and relieving the disease, such as by causing regression or restoring or repairing a lost, missing, or defective function, or stimulating an inefficient process.

[0048] As used herein, the term "activity" refers to the biological function of an enzyme or a protein that can be inhibited, reduced, or interfered by various means.

[0049] As used herein, the term "enzymatic activity" refers to one or more of physiological, catalytic, regulatory, or enzymatic activities associated with an enzyme.

[0050] As used herein, the term "effective amount" means the total amount of each inhibitor of the present invention that is sufficient to show a meaningful patient benefit, i.e., healing of tendinopathy or increase in the rate of healing and amelioration of symptoms.

I. The Components of Tendon and Cartilage

[0051] Tendon is composed of an ordered heirarchy of type I collagen, which is interspersed with small amounts of type III collagen, fibroblast-like tenocytes, and chondrocyte-like fibrochondrocytes. The type I collagen is packed into dense collagen fibers, which provide the tendon with strength and stiffness while it is stretched and pulled by the attached muscles. In contrast, cartilage is composed of type Il collagen and proteoglycans, and is designed to withstand compression, not tension. Fibrocartilage is a mixture of tendon and cartilage tissues, and is primarily found at the interface between tendon and bone. The formation of cartilage and fibrocartilage within a tendon is harmful to tendon function because it disrupts the closely packed type I collagen fibers and reduces the tensile strength of the tendon. Accordingly, the methods of the invention may be employed to prevent or reduce the formation of cartilage and/or fibrocartilage in damaged or injured tendon tissue.

[0052] One indication of cartilage formation in tendon is the presence of increased levels of proteoglycans in the tissue, often detected by the presence of the glycosaminoglycan (GAG) side chains of these proteoglycans. Proteoglycans are a family of glycoproteins characterized by a core protein that has one or more linear GAG chains, which are made up of repeating disaccharide units. Chondroitin sulfate proteoglycans, such as aggrecan and versican, are cartilage-specific. Aggrecan is the major structural component of cartilage and is responsible for the compressive strength and elasticity of cartilage. The hydration and aggregation of aggrecan molecules create a viscous gel that absorbs compressive load in cartilage tissues. Watanabe et al., J. Biochem 124(4):687-93 (1998). Other proteoglycans present in cartilage tissue include heparan sulfate proteoglycans, such as, for example, syndecan 3 (syn-3). Kirn-Safran et al., Birth Defects Res 72(Part C):69- 88 (2004). Ii. Molecules that Promote Cartilage and Fibrocartilage Formation

[0053] The invention is based, in part, on the discoverythat the expression of cartilage-specific genes (e.g., aggrecan, versican, and type Il collagen) is increased in injured tendons. This result, using gene expression profiles obtained in a rat tendon overuse model described in Soslowsky et al., J Shoulder Elbow Surg. 9:79- 84 (2000), suggests that the injured tendon is converted into fibrocartilage as a result of the overuse.

[0054] The invention is also based, in part, on the discovery that the expression of enzymes involved in the synthesis of glycosaminoglycan (GAG) sidechains of cartilage-specific proteoglycans is increased in overused tendons. The discovery that cartilage-specific enzymes are upregulated in damaged tendons provides a mechanism for the accumulation of GAGs observed in severely damaged tendons. Khan et al., Sports Med 27:393-408 (1999) and Tallon et al., Med ScI Sports Exerc 33(12):1983-90 (2001 ). These increased GAG levels indicate that levels of proteoglycans such as aggrecan, versican, and syn-3 are increased in the damaged tendons. Thus, tendinopathy can be treated by preventing the accumulation of cartilage-specific proteoglycans by reducing the activity of the enzymes responsible for producing these proteoglycans. A. Molecules that Promote Cartilage Formation are Upregulated in Tendinopathy

[0055] The Affymetrix GeneChip^® system was used to identify differences in gene expression between normal tendon and tendon subjected to an overuse protocol. The gene expression profiles yielded more than 400 genes that are differentially regulated in the supraspinatus tendon after overuse. By 4 weeks of overuse, 107 genes were upregulated and 27 genes were down-regulated. Some of the most highly upregulated genes are cartilage-specific genes, including collagen type 2 alpha-1 chain (Col2a1), Sox9, versican, and aggrecan. Other upregulated genes include those involved in the formation of GAG sidechains on cartilage proteoglycans, including CS-GalNAcT-1 , GalNT-1 , and Hs3st1. Based on these results, it is likely that these molecules themselves or other molecules responsible for the initiation or maintenance of activity of these molecules or expression of the genes encoding these molecules may be involved in the formation of cartilage and/or fibrocartilage in overused tendons. These molecules may include enzymes, transcription factors, and signal transduction factors, as listed below:

1. Enzymes

[0056] One method of inhibiting the production of cartilage or fibrocartilage in tendon tissue is to inhibit the expression or activity of the enzymes involved in proteoglycan synthesis. For a complete list of enzymes involved in proteoglycan GAG chain synthesis, see Silbert et al., IUBMB Life 54:177-186 (2002) and Sugahara et al., IUBMB Life 54:163-175 (2002). These enzymes include proteins whose expression is increased in overused tendons (Example 1). Accordingly, one embodiment of the invention involves inhibiting the expression or activity of any one of the following enzymes:

[0057] CS-GalNAcT-1 and CS-GalNAcT-2 (EC 2.4.1.174 and EC 2.4.1.175), involved in the initiation and elongation of chondroitin sulfate GAG sidechains. CS- GalNAcT-1 is involved in the initiation and elongation of chondroitin GAGs, while CS-GalNAcT-2 is involved in the elongation of those same chondroitin GAGs. For a detained description of these enzymes, please see Uyama et al., J. Biol. Chem, 277(11):8841 -8846 (2002); Gotoh et al., J. Biol. Chem. 277(41 ):38189-38196 (2002); Sato et al. J. Biol. Chem. 278(5) :3063-3071 (2003); Uyama et al., J. Biol. Chem. 278(5) :3072-3078 (2003).

[0058] UDP-D-xylose:core protein β-D-xylosyltransferase (EC 2.4.2.269), involved in the addition of the initial XyI moiety onto a proteoglycan core protein, one of the first steps of GAG sidechain synthesis;

[0059] GaI transferases (EC 2.4.1.133 and EC 2.4.1.134), involved in the addition of galactose residues onto the XyI moiety of new GAG sidechains;

[0060] GIcA transferases (EC 2.4.1.135, EC 2..4.1.226, and EC 2.4.1.225), involved in the addition of GIcA moeties onto the galactose residue of new GAG sidechains;

[0061] GIcNAc transferases (EC 2.4.1.223 and EC 2.4.1.224), involved in the addition of GIcNAc moities to the GaINAc moities of chondroitin sulfate GAGs added by CS-GaINAcT enzymes;

[0062] Sulfotransferases (EC 2.8.2.5 and EC 2.8.2.1), involved in the transfer of sulfate residues on chondroitin sulfate GAGs; [0063] Chondroitin sulfate synthases (EC 3.1.6.4 and EC 3.1.6.12), involved in the initial addition of sulfate residues to chondroitin sulfate GAGs;

[0064] GalNT-1 (E.C.2.4.1.41) catalyzes the first step in formation of O- linked oligosaccharides side-chains on glycoproteins. White et al., J Biol Chem 270(41 ):24156-65 (1995); and

[0065] Hs3st1 (EC 2.8.2.23) catalyzes the addition of sulfate residues onto heparan sulfate GAGs. Shworak et al., J Biol Chem 272(44):28008-19 (1997)

[0066] Inhibiting the activity of any one of these enzymes, either directly or indirectly, will reduce the formation of proteoglycans. Methods for inhibition of these enzymes are discussed below. 2. Gene Expression

[0067] An alternative method for preventing the formation of cartilage and/or fibrocartilage is to prevent the expression of cartilage-specific proteins in the damaged tendon tissue, either directly or indirectly. For example, the expression of versican or aggrecan, cartilage-specific proteoglycans, may be reduced by a number of methods, including preventing the transcription or translation of the genes encoding aggrecan or versican, or preventing the expression or function of transcription factors involved in the expression of the aggrecan or versican genes, which would have the result of preventing the transcription of these genes.

[0068] It is important when practicing the invention to ensure that the inhibition of cartilage-specific factors is localized to the damaged tendon, because many cartilage-specific factors, including proteoglycans and type Il collagen, are important elements of functional articular cartilage and other tissues. [0069] One method for inhibiting the formation of cartilage and/or fibrocartilage in tendon tissue is to inhibit the activity or expression of non- enzymatic proteins involved in the expression of cartilage structural proteins. By inhibiting the expression or activity of these genes, one can indirectly inhibit the expression of the proteins that make up the cartilage and/or fibrocartilage tissue. Proteins involved in the expression of cartilage structural proteins include, but are not limited to, Sox9.

[0070] Other methods for inhibiting the formation of cartilage or fibrocartilage in tendon tissue involve directly inhibiting the expression (as opposed to the activity) of major cartilage structural proteins, such as, for example, aggrecan, versican, and type Il collagen. The following genes were found to be upregulated in damaged tendon tissue and are linked to cartilage formation. a) Sox9

[0071] Sox9 (sry-type high mobility-group box 9) is a transcription factor involved in the development of cartilage. Sox9 is expressed in chondrocytes, coincident with the expression of the collagen alphal (II) gene (Col2a1). Sox9 regulates chondrogenesis by activating or enhancing the transcription of genes that express cartilage structure proteins such as type Il collagen. Shum et al., Arthritis Res. 4(2):94-106 (2002); Bi et al., Nat. Genet. 22(1):85-9 (1999). b) Col2a1

[0072] Col2a1 encodes the alpha 1 chain of type Il collagen, a major component of cartilage. Cheah et al., Proc Natl Acad Sci U S A 82(9):2555-9 (1985). c) Agc1

[0073] Agc1 encodes the core protein of the aggrecan proteoglycan, Doege et al., Extracellular Matrix Genes (Sandel, L J.; Boyd, C. D., eds.) Academic Press (New York) 137-152 (1990). As used herein, the term "aggrecan" refers to a large proteoglycan specific to cartilage tissues. Aggrecan is composed of a protein core coated with numerous chondroitin sulfate GAG moities. Aggrecan is is a major structural component of cartilage and is able to bind water and form a viscous gel- like substance. This hydration provides cartilage tissue with its elasticity and compressive strength. d) Cspg2

[0074] Cspg2 encodes the core protein of the versican proteoglycan. Versican is another large proteoglycan with numerous chondroitin sulfate sidechains. Like aggrecan, versican binds water and forms a viscous gel that increases the tissue's strength and elasticity. Knudson et al., Sem Cell Dev Biol 12:69-78 (2001).

B. Treatment of Tendinopathy by Inhibition of Molecules That Promote Cartilage Formation

[0075] The invention provides methods for treatment or prevention of tendinopathy by reducing the expression and/or activity of the molecules listed above. The invention further provides methods of administering an inhibitor of the molecules listed above to treat or prevent tendinopathy. 1. Treatment Strategy

[0076] The instant invention is based, in part, on the discovery that the expression levels of the genes encoding aggrecan, versican Sox9, type Il collagen, CS-GalNAcT-1 , GalNT-1 , and HS3st1 are increased significantly in overused tendon tissues when compared to control tissues (Figures 1-7). This result indicates that the increased expression of enzymes or other molecules involved in the proteoglycan synthesis pathway leads to the conversion of injured tendon tissue into cartilage and/or fibrocartilage. Therefore, in one embodiment, the invention includes methods for treating tendinopathy comprising reducing the formation of cartilage and/or fibrocartilage in tendon tissue by inhibiting the activity, expression, or accumulation of enzymes or other molecules involved in the synthesis of cartilage and/or fibrocartilage.

[0077] Inhibitors that can block the activity of the enzymes or other molecules involved in cartilage or fibrocartilage formation in tendon are useful in the invention. Inhibitors useful in the methods of the invention are optionally glycosylated, pegylated, or linked to another nonproteinaceous polymer. Inhibitors may be modified to have an altered glycosylation pattern (i.e., altered from the original or native glycosylation pattern). As used herein, "altered" means having one or more carbohydrate moieties added or deleted, and/or having one or more glycosylation sites added or deleted as compared to the original inhibitor. Addition of glycosylation sites to the inhibitors may be accomplished by altering the amino acid sequence to contain glycosylation site consensus sequences well known in the art. Another means of increasing the number of carbohydrate moieties is by chemical or enzymatic coupling of glycosides to the amino acid residues of the inhibitor. These methods are described in WO 87/05330, and in Aplin et al., Crit Rev Biochem 22:259-306 (1981 ). Removal of any carbohydrate moieties present on the substrate may be accomplished chemically or enzymatically as described by Sojar et al., Arch Biochem Biophys 259:52-57 (1987); Edge et al., Anal Biochem 118:131 -137 (1981); and by Thotakura et al., Meth Enzymol 138:350-359 (1987).

[0078] Proteinaceous and nonproteinaceous inhibitors including, for example, peptides, small molecules and nucleic acids may also be used in the methods of the invention. a) Peptides and Small Molecules

[0079] Inhibitors useful in the methods of the invention include small organic peptides and molecules and small inorganic molecules. These peptides and small molecules include synthetic and purified naturally occurring inhibitors. Methods to identify peptides and small molecules that specifically target a protein of interest are well known in the art. For example, peptide and/or small molecule libraries may be screened for inhibition of target proteins, such as, e.g., CS-GalNAcT-1 , CS- GalNAcT-2, GalNT-1 , and/or Sox9 using an assay of the target protein's enzymatic and/or biological function. In another embodiment, small molecule and/or peptide libraries may be screened in a competitive radioligand binding assay of a target protein, such as CS-GalNAcT-1 , CS-GalNAcT-2, GalNT-1 , and/or Sox9, with their substrates or ligands.

[0080] Alternatively, fluorescent resonance energy transfer (FRET)-based assays, such as the Time-Resolved FRET (TR-FRET) assay, may be used to identify an inhibitor. In an exemplary embodiment, the core protein of a proteoglycan is labeled with a His or GST tag and an anti-His or GST antibody coupled to Europium, a fluorophore, is used to label the protein. Alternatively, the core protein is labeled non-specifically with Europium on lysine or cysteine residues. The donor sugar molecule is labeled with Cy5, a fluorescent dye. A list of donor and acceptor molecules useful in this assay can be found in Table I of Uyama et al., J Biol Chem 278(5):3072-78 (2003).

[0081] The acceptor and donor molecules are combined in different ratios with and without the target enzyme. The TR-FRET assay is performed by exciting the system at 340 nm, measuring the emission of the Europium and Cy5 at 615 and 665 nm, respectively, and calculating the ratio of Cy5 emission to Europium emission.

[0082] When there is no enzyme present, the donor and acceptor molecules do not come into close proximity to each other and there is no quenching of the individual fluorescence of the Cy5 and Europium molecules, resulting in a baseline level ratio. When the target enzyme is added to the assay system, the donor sugar molecule is transferred to the acceptor protein, and the fluorescence of the system is quenched. The ratio of Cy5 to Europium fluorescence increases with the amount of sugar molecule transferred up to a maximum.

[0083] To identify inhibitors of the target enzyme, peptides or small molecules are added to the assay system. When a test compound is able to inhibit the transfer of the sugar to the protein, the quenching of the fluorescence decreases. If the test compound is able to inhibit 100% of the transfer activity of the enzyme, the ratio of Cy5 emission to Europium emission is at baseline levels. Compounds that inhibit at least 50% of enzyme activity in this assay are then evaluated in cell-based systems to assess their ability to reduce GAG synthesis (described below). [0084] The invention also provides the use of additional screening assays, e.g. secondary and tertiary assays, to further identify the effect of such molecules on tendon morphology, for example, using assays described in detail above. b) Dominant Negative Mutants

[0085] In certain embodiments of the invention, mutant CS-GalNAcT-1 , CS- GalNAcT-2, GalNT-1 , Hs3st1 , ot Sox9 proteins may be used as inhibitors in the methods of the invention. For example, a naturally occurring variant, or an engineered homolog having dominant negative effect on the activity of molecules mentioned above, both in vivo and in vitro, may be used in the methods of the invention. c) Nucleic Acids

[0086] Nucleic acids that that can block the activity of target molecules listed above, are useful in this invention. Such inhibitors may encode proteins that interact with, for example, CS-GalNAcT-1 , CS-GalNAcT-2, GalNT-1 , Hs3st1 , or Sox9. Alternatively, such inhibitors may encode proteins that can interact with substrates or ligands of the target molecule (such as GaINAc) and may be effective in the methods of the invention if the encoded proteins block the binding of the target molecule to its substrate or ligand or if they block the activity of the substrate or ligand after binding of the target molecule. Inhibitors, of course, may encode proteins that interact with the target molecule and its substrate or ligand at the same time. Such nucleic acids can be used to express, for example, CS-GaINAcT- 1 , CS-GalNAcT-2, Hs3st1 , GalNT-1 , or Sox9 inhibitors for use in the methods of the invention. [0087] The methods of the invention also encompass the use of interfering RNA molecules ("RNAi"), including, but not limited to, short interfering RNAs (siRNA), short hairpin RNAs (shRNA), and short double stranded RNA (sdsRNA), to reduce the expression of any of the molecules listed above or their protein binding partners. RNAi can be initiated by introducing nucleic acid molecules, e.g. synthetic short interfering siRNAs or RNA interfering agents, to inhibit or silence the expression of target genes. See, for example, U.S. Patent Pub. Nos. 2003/0153519 and 2003/01674901 , and U.S. Patent Nos. 6,506,559, and 6,573,099.

[0088] The siRNA may be chemically synthesized, produced by in vitro transcription, or produced within a host cell. Typically, a siRNA is at least 15-50 nucleotides long, e.g., 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The siRNA may be a double stranded RNA (dsRNA) of about 15 to about 40 nucleotides in length, for example, about 15 to about 28 nucleotides in length, including about 19, 20, 21 , or 22 nucleotides in length, and may contain a 3' and/or 5' overhand on each strand having a length of about 0, 1 , 2, 3, 4, 5, or 6 nucleotides. The siRNA may inhibit a target gene by transcriptional silencing. Preferably the siRNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA.

[0089] siRNAs used in the methods of the invention also include small hairpin RNAs (shRNAs). shRNAs are composed of a short (e.g. about 19 to about 25 nucleotide) antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow. These shRNAs may be contained in plasmids and viral vectors.

[0090] The targeted region of the siRNA molecules may be selected from a given target sequence. For example, nucleotide sequences can begin from about 25-100 nucleotides downstream of the start codon. Nucleotide sequences can contain 5' or 3' untranslated regions, as well as regions near the start codon. Methods for the design and preparation of siNRA molecules are well known in the art, including a variety of rules for selecting sequences as RNAi reagents. See, e.g., Boese et al., Methods Enzymol 392:73-96 (2005).

[0091] siRNA may be produced using standard techniques as described in Hannon et al., Nature 418:244-251 (2002); McManus et al., Nat Reviews 3:737-747 (2002); Heasman et al., Dev Biol 243:209-214 (2002); Stein et al., J Clin Invest, 108:641 -644 (2001); and Zamore et al., Nat Struct Biol 8 (9): 746-750 (2001 ). Preferred siRNAs are 5' phosphorylated.

[0092] siRNA inhibitors can be used to target, for example, CS-GalNAcT-1 , CS-GalNAcT-2, GalNT-1 , Hs3st1 , Sox9, Agd , Cspgi , or Col2a1 , or other molecules listed in above, as well as their substrates or ligands.

[0093] The nucleic acids may be obtained, isolated, and/or purified from their natural environment, in substantially pure or homogeneous form. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include, e.g., Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. For other cells suitable for producing proteins from nucleic acids, see Gene Expression Systems, Eds. Fernandez et al., Academic Press (1999). RNAi molecules may be chemically synthesized or expressed from DNA sequences encoded the siRNA or shRNA sequences.

[0094] For production of dominant negative mutants of the molecules listed above, suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, selection or marker genes and other sequences as appropriate. Vectors may be plasmids or viral, e.g., phage, or phagemid, as appropriate. For further details see, e.g., Molecular Cloning: A Laboratory Manual, Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press (1989). Many known techniques and protocols for manipulation of nucleic acid, for example, in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Eds. Ausubel et al., 2nd ed., John Wiley & Sons (1992).

[0095] A nucleic acid can be fused to other sequences encoding additional polypeptide sequences, for example, sequences that function as a marker or reporter. Examples of marker or reporter genes include lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (responsible for neomycin (G418) resistance), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding -galactosidase), xanthine guanine phosphoribosyltransferase (XGPRT), luciferase. Many others are well known in the art.

2. Inhibition of Enzymatic Activity

[0096] In particular embodiments of the invention, methods for treating tendinopathy comprise reducing, inhibiting or downregulating the activity, of enzymes involved in the biosynthesis of proteoglycans, such as those involved in the addition of chondroitin sulfate GAGs to aggrecan and/or versican. These enzymes include, e.g., CS-GalNAcT-1 , CS-GalNAcT-2, UDP-D-xylose:core protein β-D-xylosyltransferase, GaI transferases, GIcA transferases, GIcNAc transferases, sulfotransferases, chondroitin sulfate synthases, Hs3st1 , and GalNT-1.

[0097] In an exemplary embodiment, the invention includes methods for reducing, inhibiting or downregulating the activity of CS-GalNAcT-1 , CS-GaINAcT- 2, GalNT-1 , and/or Hs3st1 enzymes in damaged tendons. In other embodiments, the invention includes methods for reducing, inhibiting, or downregulating XyI transferase, GaI transferase, GIcA transferase, GaINAc transferase, GIcNAc transferase, sulfotransferase, or chondroitin sulfate synthase activity in damaged tendons. The peptide or small molecule inhibitors described above may be used to practice these methods. a) Identifying Inhibitors of Enzyme Activity

[0098] The invention further comprises methods of identifying and evaluating the efficacy of agents that could act as inhibitors of CS-GalNAcT-1 , CS-GalNAcT-2, GalNT-1 , Hs3st1 , XyI transferase, GaI transferase, GIcA transferase, GaINAc transferase, GIcNAc transferase, sulfotransferase, and/or chondroitin sulfate synthase in damaged tendon tissue. [0099] Such inhibitors block or reduce the activity of the enzymes involved in cartilage or fibrocartilage formation in tendon. These inhibitors include modified soluble substrates, proteins, small molecules, and nucleic acids.

[0100] Methods of identifying an agent for treating tendinopathy involve assaying the effect of each individual agent on, enzyme activity, protein-target interaction, or protein-protein interaction of the enzymes listed above. Various in vivo or in vitro assays may be used to determine the efficacy of an inhibitor in treating tendinopathy. In one exemplary embodiment, small molecules are evaluated in vitro for their potential therapeutic utility by assaying their ability to affect, for example, enzyme activity, target binding, or protein-protein interactions of the enzymes listed above by a method like high throughput screening (HTS).

[0101] In a typical HTS assay the inhibitory effect of a candidate compound on, for example, enzyme activity, ligand-receptor binding, and the like, can be measured by comparing the endpoint of the assay in the presence of a known concentration of the candidate to a reference which is performed in the absence of the candidate and/or in the presence of a known inhibitor compound. Generally, conventional dye, fluorescent, or radio active molecules are used to monitor the effect of the candidate compound. Thus, for example, a candidate compound can be identified which inhibits the binding of a ligand and its receptor, or which inhibits enzyme activity, decreasing the turnover of the enzymatic process.

[0102] An exemplary embodiment of the present invention provides an in vitro method of identifying an agent for treating tendinopathy comprising: (1) providing a sample of at least one sample component selected from the group consisting of CS-GalNAcT-1 , CS-GalNAcT-2, GalNT-1 , and Hs3st1 ; (2) combining the sample component with a test agent; (3) measuring the activity of the sample component in response to the test agent; and (4) determining whether the test agent inhibits the activity of the sample component.

[0103] In one exemplary embodiment, an in vitro method of identifying an agent for treating tendinopathy involves treating a cartilage explant, a primary chondrocyte pellet culture, or a chondrocyte cell line with a BMP-2 protein or an adenovirus expressing a BMP-2 protein. The BMP-2 stimulates ECM and GAG synthesis. A test compound is then added to the culture and the effect of the inhibitor on GAG synthesis is evaluated by measurement of 35S incorporation into the GAG sidechains and/or by the DMMB assay. The effect of the test compound will be compared to the appropriate vehicle controls.

[0104] An in vivo assay of a potential enzyme inhibitor may comprise: (1 ) administering a test inhibitor repeatedly to a mammal (e.g., an Sprague-Dawley rat) for a period of at least 2, 4, 6, or 8 weeks; (2) subjecting the mammal to a downhill running protocol at 17 m/min for 1 hour/day, 5 days/week (Example 1); and (3) determining the effect of the inhibitor on the supraspinatus tendon by DMMB or AB assay or by gene expression profiling, wherein a slowing of tendon degeneration (e.g., cellular and morphological abnormalities) is considered to be attributable to the inhibitor and indicates that the inhibitor is effective for treatment of a tendon degenerative disorder.

[0105] It will be understood that a test inhibitor useful in the methods of the invention may be evaluated in one or more animal models of tendon degenerative disorders and/or in humans. [0106] Various assays for measuring enzyme activity in the presence of an inhibitor in vivo and in vitro are known in the art. Examples of some of the more frequently used assays include spectrophotometry, spectrofluorimetry, circular dichroism, automated spectrophotometric and spectrofiuorimetric procedures, coupled assays, automatic titration of acid or base, radioactive procedures, label- free optical detection, and enzyme inhibition assay. In exemplary embodiments, the enzyme activity of CS-GalNAcT-1 and/or CSGalNAcT-2 is measured as described in Uyama et al., J Biol Chem 277(11):8841 -46 (2002). In another exemplary embodiment, the enzyme activity of GalNT-1 is measured as described in White et al., J Biol Chem 270 (41 ):24156-65 (1995).

[0107] Enzyme inhibitors useful in the methods of the invention, for example, may interact with the enzymes they inhibit. Alternatively, inhibitors may interact with an enzyme substrate (such as GaINAc) or other binding partners, for example. Inhibitors may reduce or and may be effective in the invention if they block the binding of the enzyme to its substrate and/or if they block the activity of the substrate after binding of the enzyme. Inhibitors, of course, may interact with both the enzyme and a second factor, such as its substrate. In this regard, CS- GalNAcT-1 , CS-GalNAcT-2, GalNT-1 , and/or Hs3st1 inhibitors include, for example, modified soluble substrates, other proteins (including those that bind to the enzyme and/or an enzyme substrate), modified forms of the enzyme or fragments thereof, propeptides, peptides, and mimetics of all of these inhibitors.

[0108] An enzyme inhibitor may, for example, be a direct enzyme inhibitor; bind to and neutralize the activity of the enzyme; decrease the enzyme expression levels; affect stability or conversion of the precursor molecule to the active, mature form; interfere with the binding of the enzyme to one or more of its substrates; or it may interfere with intracellular functions of the enzyme.

3. Inhibition of Transcription Factor Activity

[0109] Inhibitors of transcription factors useful in the methods of the invention may inhibit or reduce the activity of factors directly by binding or indirectly by interfering with their protein-protein interactions and/or binding properties. In this regard, inhibitors of Sox9 may include modified soluble ligands or target molecules, small molecules, and nucleic acids.

[0110] Assays for measuring Sox9 activity in the presence of an inhibitor in vivo and in vitro is known in the art. Examples of some of the more frequently used assays for Sox9 inhibitors include the yeast two-hybrid system, luciferase reporter gene; PCR assays for determining the expression levels of chondrocyte- specific genes (e.g., Col2a1 , Agd ) in the presence of a Sox9 inhibitor, western-blot analysis of Sox9 or chondrocyte-specific genes, transient transfection, chloramphenicol acetyltransferase (CAT) assays, and northern-blot analyses.

[0111] Various protein-protein or protein-nucleic acid interaction assays, based on fluorescence resonance energy transfer (FRET), may be utilized to assay the effect of inhibitors on Sox9 interactions with its binding partners. For example, one can use AlphaScreen™ amplified luminescence proximity homogeneous assay (from PerkinElmer®) or Bioluminesence Resonance Energy Transfer (BRET™ from PerkinElmer®) may be employed to perform an in vitro assay for inhibitors that interfere with Sox9 binding interactions.

[0112] In one embodiment, the present invention provides a method for an in vitro assay for identifying an agent for treating tendinopathy comprising: (1) providing a target molecule listed above fused to a donor fluorogenic molecule; combining the target molecule with a test agent; (3) adding the binding partner of the target molecule fused to an acceptor fluorescent molecule; (4) measuring the fluorescence energy transfer levels between the target molecule and its binding partner; and (5) determining whether the test agent inhibits the interaction of the target molecule with its binding partner.

[0113] An inhibitor can interfere with binding of a target molecule to its binding partner and subsequently affect the FRET levels between the two. Accordingly, FRET measurements can be used to identify inhibitors of various efficiency. Once an inhibitor of a target molecule is identified in vitro, further in vivo testing can be performed to determine the efficacy and applicability of the inhibitor in preventing the formation of cartilage and fibrocartilage in tendon. 4. Inhibition of Protein Expression

[0114] In another embodiment, methods for treating tendinopathy comprise reducing, inhibiting or downregulating the expression and/or accumulation of genes such as CS-GalNAcT-1 , CS-GalNAcT-2, GalNT-1 , Hs3st1 , Col2a1 , Cspgi , Agc1 , and/or Sox9, and their encoded polypeptides or proteins. Compositions useful for inhibiting the expression of these genes include, e.g., interfering RNA molecules, which are described in Section N(B)(I )(c) above.

[0115] These inhibitors may be administered in any manner, but in particular embodiments, DNA expressing an RNAi inhibitor is incorporated into an adenovirus vector, which is then injected into a patient in need of tendon repair. In exemplary embodiments, the nucleotide sequence encoding the RNAi is positioned downstream from a polll promoter, as described in Wahdwa et al., Curr Opin MoI Ther 6(4):367-72 (2004). Methods for inserting siRNA and shRNA into adenovirus vectors are well known in the art and are described in, for example, Krom et al., BMC Biotech 6(11):[e-published ahead of print]. Alternatively, the RNAi inhibitor is injected directly into the injured tendon tissue and transported into cells via electroporation, as described in Schiffelers et al., Arthritis & Rheumatism 52(4):1314-18 (2005).

5. Tendinopathy Conditions

[0116] The methods of the invention may be used to treat or prevent a tendinopathy in any mammal in need of such treatment, including humans, primates, monkeys, rodents, sheep, rabbits, dogs, guinea pigs, horses, cows, and cats. The disorders that may be treated or prevented include, for example, tendinitis, tendonitis, tendinosis, paratendinitis, tenocynovitis, tendon overuse injuries, tendon trauma, perintendinitis, paratenonitis, and any other conditions in the family of "tendinopathy" conditions.

[0117] Tendinopathy may be caused by overuse and repeated movements, a sudden injury (ranging from mild to severe), gradual degeneration, or aging. Most tendon injuries consist of a slow-healing series of microtears (tendinosis) that weaken a tendon, often causing pain, stiffness, and loss of strength. Tendinopathies usually require several weeks of treatment, reduced or modified activity, and rest. Returning the injured tendon to use too soon can lead to more tendon damage, rendering the damaged tendon more susceptible to tears or rupture. Accordingly, the disorders treated or prevented by the present invention include tendon degenerative disorders, both acute and chronic, that are associated with overuse, sport- or accident-related injuries and trauma, nutritional deficiencies, and advanced age.

[0118] One example of a tendinopathy is lateral epicondylitis, also known as "tennis elbow," a well-known sports medicine and orthopedic disorder. The pathology underlying the disorder is related to overuse and microtearing of the extensor carpi radialis brevis tendon at the elbow. The body attempts to repair these microtears but in many cases the healing process is incomplete. Pathologic specimens of patients undergoing surgery for chronic lateral epicondylitis reveal a disorganized angiofibroblastic dysplasia in the damaged tendon. This incomplete attempt at repair results in degenerated, immature, and vascularized tissue. Incompletely repaired tissue is weaker than normal tendon tissue and lacks the strength to function normally. This weakened tissue also limits the patient by causing pain and negatively impacting quality of life. Similar incomplete repair may be present in other types of tendon related injuries or damage, such as patellar tendinitis (Jumper's Knee), Achilles tendinitis (common in runners), and rotator cuff tendinitis (commonly seen in "overhead" athletes such as baseball pitchers).

[0119] Tendinopathy may also be caused by, for example, advanced age, poor diet, sporting activities, trauma, injuries, or drugs such as pefloxacin. For example, prostaglandin E1 (PGE1)-, prostaglandine E2 (PGE2)- or pefloxacin- induced tendinopathy is a well established animal model of human tendinopathy. In one embodiment, the invention provides methods to treat or prevent drug- induced tendinopathy such as pefloxacin-induced tendinopathy in an individual. In other exemplary embodiments, the invention provides methods to treat or prevent tendinopathy induced by overuse, sport- or accident-related injuries and trauma, nutritional deficiencies, and advanced age.

[0120] The present invention further provides methods to treat or prevent tendon degenerative disorder, slow tendon deterioration, restore tendon quality, maintain tendon health, treat or prevent hypoxic degeneration of tendon tissues, treat or prevent hyaline degeneration of tendon tissues, treat or prevent mucoid or myxoid degeneration of tendon tissues, treat or prevent fibrinoid degeneration of tendon tissues, treat or prevent lipoid degeneration of tendon tissues, treat or prevent calcification of tendon tissues, treat or prevent fibrocartilaginous and bony metaplasia of tendon tissues, maintain microstructural integrity of tendon, maintain fiber structure in tendon, maintain fiber arrangement in tendon, maintain normal tendon vascularity, maintain normal cellularity in tendon tissues, restore normal ECM content in tendon tissues, reduce the pathological accumulation of GAG in tendon tissue; and/or maintain tendon mass, tensile strength, and/or flexibility quality by administering to a mammal an inhibitor of the activity and/or expression of molecules such as CS-GalNAcT-1 , CS-GalNAcT-2, GalNT-1 , XyI transferase, GaI transferase, GIcA transferase, GaINAc transferase, GIcNAc transferase, sulfotransferase, chondroitin sulfate synthase, Col2a1 , aggrecan, versican, Hs3st1 , and/or Sox9 in an amount effective to prevent or reduce the activity and/or expression of such molecules in tendinopathic tissues.

6. Assessing Treatment and Animal Models

[0121] The methods of the invention may be used to treat tendinopathy or microdefects in tendon tissues. The tendon quality following or during a treatment can be determined, for example, by assessing microstructural integrity of tendon, tendon collagen stainability, variation in tendon tissue cellularity, tendon vascularization, and/or GAG content. Methods for evaluating tendon health following or during a treatment are known in the art and include, but are not limited to, magnetic resonance imaging (MRI), ultrasonography, and functional and/or pain assessments. Alternatively, tendon health may be measured by evaluating the levels of GAG in tendon tissue.

[0122] One assay for determining the GAG content of tendon is 1 ,9- dimethylmethylene blue (DMMB) assay for the measurement of sulfated glycosaminoglycan (GAG) concentrations in tendon. Another assay for determining the GAG content of tendon is histological Alcian blue (AB) staining which can be used to detect increased amount of GAG in tendon tissues. III. Methods of Treatment and Pharmaceutical Compositions

[0123] Inhibitors useful in the methods of the invention may be administered locally or systemically via topical, oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, implant, or transdermal means.

[0124] In exemplary embodiments, administration of an inhibitor to an individual may also be accomplished by means of gene therapy, wherein a nucleic acid sequence encoding the inhibitor is administered to the patient in vivo. In an exemplary embodiments, a nucleic acid comprising a promoter sequence and a sequence encoding a nucleic acid inhibitor useful in the methods of the invention is inserted into an adenovirus expression vector. The adenovirus is then injected directly into the tendon of a patient, where the nucleic acid inhibitor is expressed. Examples of adenovirus-mediated delivery of nucleic acids to tendon tissue are described in, e.g., Mehta et al., J Hand Surg 30A(1):136-41 (2005); Lou et al., J Orthoped Res 19:1199-1202 (2001), and Lou, Clin Orthopaed ReI Res 379S:S252- 55 (2000).

[0125] In other exemplary embodiments, the nucleic acid inhibitor is incorporated into other viral expression vectors, such as, for example, lentiviruses, herpesviruses, and adeno-associated-viruses. The appropriate vector may be chosen by evaluating the in vitro infectivity of each type of virus in primary tendon cells.

[0126] Alternatively, a nucleic acid inhibitor is injected into tendon tissue directly, and transferred into cells by electroporation, as described in Schiffelers et al., Arthritis & Rheumatism 52(4):1314-18 (2005).

[0127] In other exemplary embodiments, peptide or small molecule inhibitors useful in the methods of the invention may be coated onto transdermal patches and applied directly to the skin covering the tendon tissue, as described in Paoloni et al., J Bone Joint Surg 86A(5):916-22 (2004).

[0128] Methods of administration of molecules for the treatment of tendinopathy are known in the art. "Administration" is not limited to any particular delivery system and may include, without limitation, parenteral (including subcutaneous, intravenous, intramedullary, intraarticular, intramuscular, intracavity, or intraperitoneal injection) rectal, topical, transdermal, or oral (for example, in capsules, suspensions, or tablets). Administration to an individual may occur locally or systemically in a single dose or in continuous or intermittent repeat administrations, and in any of a variety of physiologically acceptable salt forms, and/or with an acceptable pharmaceutical carrier and/or additive as part of a pharmaceutical composition (described earlier). Physiologically acceptable salt forms and standard pharmaceutical formulation techniques and excipients are well known to persons skilled in the art. See, e.g., Physicians' Desk Reference (PDR) 2003, 57th ed., Medical Economics Company, 2002; and Remington: The Science and Practice of Pharmacy, eds. Gennado et al., 20th ed, Lippincott, Williams & Wilkins, 2000).

[0129] Inhibitors useful in the methods of the invention may be administered at a dosage from about 1 μg/kg to about 20 mg/kg, depending on the severity of the symptoms and the progression of the disease. The appropriate effective dose is selected by a treating clinician from the following ranges: about 1 μg/kg to about 20 mg/kg, about 1 μg/kg to about 10 mg/kg, about 1 μg/kg to about 1 mg/kg, about 10 μg/kg to about 1 mg/kg, about 10 μg/kg to about 100 μg/kg, about 100 μg to about 1 mg/kg, and about 500 μg/kg to about 1 mg/kg, for example.

[0130] In another exemplary embodiment, an inhibitor is administered repeatedly for a period of at least 2, 4, 6, 8, 10, 12, 20, or 40 weeks or for at least 1 , 1.5, or 2 years or up to the life-time of the subject, for example for the treatment of tendinopathy. In another embodiment of the invention, an inhibitor can be administered in a single dose, for example to stimulate restoration of tendon's normal structure and composition.

[0131] Generally, inhibitors useful in the methods of the invention may be administered at a dose between 10-8 and 10-7; 10-7 and 10-6; 10-6 and 10-5; or 10-5 and 10-4 g/kg. Therapeutically effective dosages achieved in one animal model can be converted for use in another animal, including humans, using conversion factors known in the art. See, e.g., Freireich et al., Cancer Chemother Reports 50(4):219-244 (1966).

[0132] The exact dosage of an inhibitor to be used in the methods of the invention is determined empirically based on the desired outcome(s). Exemplary outcomes include: (1) tendon degenerative disorder is treated or prevented; (2) tendon deterioration is slowed; (3) quality of tendon is restored; (4) tendon health is maintained; (5) hypoxic degeneration of tendon is treated or prevented; (6) hyaline degeneration of tendon is treated or prevented; (7) mucoid or myxioid degeneration of tendon is treated or prevented; (8) fibrinoid degeneration of tendon is treated or prevented; (9) lipoid degeneration of tendon is treated or prevented; (10) calcification of tendon is treated or prevented; (11) fibrocartilaginous and bony metaplasia of tendon is treated or prevented; (12) fiber structure in tendon is maintained; (13) fiber arrangement in tendon maintained; (14) normal vascularity of tendon is maintained; (15) normal cellularity in tendon is maintained; (16) normal ECM contend in tendon is restored; and/or (17) tendon mass, tensile strength and/or flexibility quality is maintained. For example, an inhibitor is administered in an amount effective to slow tendon deterioration (e.g., loss of tendon mass, structural makeup and/or susceptibility to microtears and pain) by at least 20, 30, 40, 50, 100, 200, 300, 400, or 500%.

[0133] In some embodiments, compositions used in the methods of the invention further comprise a pharmaceutically acceptable excipient. As used herein, the phrase "pharmaceutically acceptable excipient" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances are well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions. The pharmaceutical compositions may also be included in a container, pack, or dispenser together with instructions for administration.

[0134] A pharmaceutical composition to be administered in connection with the methods of the invention should be formulated to be compatible with its intended route of administration. Examples of such formulations include crystalline protein formulations, provided naked or in combination with biodegradable polymers (e.g., PEG, PLGA).

[0135] An inhibitor useful in the methods of the invention may be administered as a pharmaceutical composition in conjunction with carrier gels and matrices or other compositions used for guided bone regeneration and/or bone substitution. Examples of such matrices include synthetic polyethylene glycol (PEG), hydroxyapatite, collagen and fibrin-based matrices, Tisseel® fibrin glue, etc.

[0136] In certain embodiments inhibitors may be administered in combination or concomitantly with other therapeutic compounds such as, e.g., NSAIDs, corticosteroids, nitric oxide, testosterone, estrogen, growth factors (e.g., BMP-12, BMP-13, and MP52).

[0137] Inhibitors useful in the methods of the invention may be coated onto or incorporated into tendon implants, matrices, and depot systems so as to promote treatment or prevention of tendon injuries. EXAMPLES

Example 1: Rat Supraspinatus Tendon Expresses Cartilage Markers With Overuse

[0138] The response of the rat supraspinatus tendon to overuse was studied at the molecular level using transcriptional profiling. A rat model of tendon overuse has previously been described, Soslowsky et al., J Shoulder Elbow Surg 9:79-84 (2000). It has been shown that inflammatory and angiogenic markers are altered in this model (Perry et al., J Shoulder Elbow Surg 14:79S-83S (2005)), but a broader approach was undertaken to understand the events surrounding the overuse injury.

[0139] Twenty-four male Sprague-Dawley rats (400-450 g) were subjected to a supraspinatus tendon (SST) overuse protocol, for 1 week (n=8), 2 weeks (n=8), and 4 weeks (n=8). The protocol consists of downhill running (10% grade) at 17 m/min for 1 hour/day, 5 days/week. An additional six rats were used as cage- activity controls (time 0). At each time point, two additional non-running rats were used as age-matched cohort controls.

[0140] At necropsy, the supraspinatus tendons from each shoulder and the patellar tendons (PT) from each knee were removed. The patellar tendon served as an internal control of the effect of exercise, because it does not show gross signs of overuse with this protocol. The harvested tendons were weighed and snap frozen in liquid nitrogen. The tissues were freeze-fractured and extracted with TRIzol reagent (Invitrogen). RNA was isolated from the aqueous phase of the extract using an RNeasy kit (QIAGEN). RNA concentrations were determined using a spectrophotometer. Transcriptional profiling to monitor the expression level of greater than 30,000 transcripts was performed with an Affymetrix rat genome 230 2.0 array. All array images were visually inspected for defects and quality. Arrays with high background, low signal intensity, or major defects were eliminated from further analysis. Signal values were determined using Gene Chip Operating System 1.0 (GCOS, Affymetrix). For each array, statistical values were normalized to a mean signal intensity value of 100. The default GCOS statistical values were used for all analysis. A gene was considered detectable if the mean expression in any tissue was greater than 100 signal units and the percentage of samples with a Present (P) call as determined by GCOS default settings was greater than or equal to 66%. Normalized signal values were transformed to the log base 10. A gene was considered to be differentially expressed if the p-value from an ANOVA test was <0.01 and the difference between running and control was at least 2 fold at any time point.

[0141] Over 400 genes were differentially regulated in the supraspinatus tendon after overuse. By 4 weeks of running 107 genes were up-regulated and 27 genes were down-regulated. Of the top up-regulated genes, many are highly expressed in cartilage tissues, including collagen type Il alpha 1 , versican, and aggrecan. These results suggested that the overused tendon was converting to a fibrocartilage phenotype. These same genes were not up-regulated in the patellar tendons of the animals that were run (see Figures 1-7). Example 2: Preparation of siRNA Inhibitor

[0142] An siRNA is selected by entering the nucleotide sequence for CS- GalNAcT-1 , GalNT-1 , or Hs3st1 into a commercial website to design specific siRNAs (e.g. sirnawizard.com or Ambion® siRNA Target Finder). Sequence selection guidelines are embedded in these tools.

[0143] The efficacy of the siRNA is measured by transfecting it into C- 20/A4 and/or C-28/I2 chondrocyte cells and monitoring the expression of CS- GalNAcT-1 and/or GalNT-1 by real-time RT-PCR. Appropriate scramble siRNA with the same nucleotide composition is used an experimental control. Example 3: In vitro Assay System for Inhibitor of Proteoglycan Synthesis

[0144] If the siRNA is capable of reducing the expression of CS-GaINAcT- 1 , GalNT-1 , or Hs3st1 in the C-20/A4 and/or C-28/I2 chondrocyte cells, it is then tested for its ability to reduce the production of proteoglycan GAG sidechains. C- 20/A4 and/or C-28/I2 chondrocyte cells are treated with BMP-2 protein or an adenovirus expressing BMP-2 to stimulate extracellular matrix and GAG synthesis. The siRNA or, alternatively, small molecule, inhibitor of GAG synthesis is added to the culture and the effect of the inhibitor is evaluated by comparing 35S incorporation into proteoglycans in the presence or absence of the inhibitor. Alternatively, the effect of the inhibitor is evaluated by comparing GAG levels in the presence or absence of the inhibitor with the DMMB assay, as described in Arai et al., Osteoarthritis and Cartilage 12:599-613 (2004).

Example 4: Protocol for Treatment of Tendinopathy by siRNA Inhibition of CS-GalNAcT-1 , GalNT-1, or Hs3st1

[0145] Tendinopathy is induced in rats either with a treadmill overuse protocol (Example 1) or local injection of PGE1 , PGE2 or Pefloxacin (300 mg/ml), five times per week for 1 week. [0146] Once an siRNA is found to reduce the activity of CS-GalNAcT-1 , GalNT-1 , or Hs3st1 in vitro, it is converted into shRNA and the shRNA sequence is cloned and inserted into an adenovirus, as described, for example, in Krom et al., BMC Biotech 6(11):[e-pubiished]. The adenovirus is used for in vivo expression of the shRNA and subsequent inhibition of the expression of CS-GalNAcT-1 and/or GalNT-1. Infection methods are optimized in primary tendon cells.

[0147] The functional adenovirus expressing the shRNA is directly injected into the injured tendon (Mehta et al., J Hand Surg 30A(1):136-41 (2005)) to locally reduce expression of the CS-GalNAcT-1 and/or GaINT- 1 genes. Scramble sequences are included as experimental controls. The amount of cartilage tissue formation in the tendons evaluated by assessing GAG levels. Histological Alcian Blue staining is used to detect increased amount of glycosaminoglycan (GAG) in the tendon tissue. DMMB reagent is used to quantify the amount of sulfated GAGs extracted from the tendon.

[0148] In vivo evaluations of the effect of inhibition of CS-GalNAcT-1 and/or GaiNT-1 on tendinopathy are determined by histology and mechanical or functional testing of tendon strength. An inhibitor is considered effective if GAG levels are significantly reduced when compared to untreated controls and/or are reduced to levels similar to normal, undamaged tendon. Alternatively, an inhibitor is considered effective if functional or mechanical tests demonstrate improved function and/or reduced pain when compared to untreated controls.

Claims

1. A method of treating tendinopathy in a patient comprising reducing the formation of cartilage-specific proteoglycans in the patient's tendon tissue.

2. The method of claim 1 , wherein the patient is a human.

3. The method of claim 1 , wherein the patient is selected from the group consisting of primates, monkeys, rodents, sheep, rabbits, dogs, guinea pigs, horses, cows, and cats.

4. The method of claim 1 , comprising reducing the activity of an enzyme involved in the synthesis of a proteoglycan selected from the group consisting of aggrecan, versican, and syndecan-3 in the patient's tendon tissue.

5. The method of claim 1 , comprising reducing the activity of chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CS-GalNAcT-1) and/or galactosamine polypeptide N-acetylgalactosaminyltransferase 1 (GalNT-1) in the patient's tendon tissue.

6. The method of claim 1 , wherein the activity is reduced by inhibiting the enzymatic activity of CS-GalNAcT-1 , CS-GalNAcT-2, Hs3st1 , and/or GalNT-1.

7. The method of claim 1 , wherein the activity is reduced by inhibiting the expression of CS-GalNAcT-1 , CS-GalNAcT-2, Hs3st1 , and/or GalNT-1 genes.

8. The method of claim 1 , comprising reducing the activity of a transcription factor involved in the production of cartilage.

9. The method of claim 8, comprising reducing the activity of Sox9.

10. The method of claim 9, wherein the activity is reduced by inhibiting the ability of Sox9 to enhance the expression of cartilage-specific genes.

11. The method of claim 8, wherein the activity is reduced by inhibiting the expression of the Sox9 gene.

12. The method of claim 1 , comprising reducing the expression of cartilage-specific proteins.

13. The method of claim 12, comprising reducing the expression of a proteoglycan selected from the group consisting of aggrecan, versican, and syndecan-3.

14. The method of claim 12, comprising reducing the expression of Col2a1.

15. The method of claim 1 , wherein the tendinopathy is tendinitis.

16. The method of claim 1 , wherein the tendinopathy is tendinosis.

17. The method of claim 1 , wherein the tendinopathy is a tendon injury.

18. A method of treating tendinopathy in a patient comprising administering an inhibitor of CS-GalNAcT-1 , CS-GalNAcT-2, GalNT-1 , Hs3st1 , and/or Sox9 to tendon tissue in a patient.

19. The method of claim 18, wherein the inhibitor reduces the enzymatic activity of CS-GalNAcT-1 and/or GalNT-1.

20. The method of claim 18, wherein the inhibitor reduces the expression of CS-GaINAcT- 1 and/or GalNT-1.

21. The method of claim 18, wherein the inhibitor is administered locally.

22. The method of claim 18, wherein the inhibitor comprises an interfering RNA molecule.

23. The method of claim 18, wherein the inhibitor comprises a small molecule.

24. A method of treating tendinopathy in a patient comprising reducing the expression of cartilage-specific proteins in the patient's tendon tissue.

25. The method of claim 24, comprising inhibiting the expression of aggrecan, versican, and/or type Il collagen.

26. The method of claim 24, comprising inhibiting the expression of Sox9.

27. The method of claim 24, comprising inhibiting the activity of Sox9.

28. A method for identifying an agent for treating tendinopathy comprising administering a test agent to a subject in need of treatment and measuring the ability of the agent to inhibit the activity of an enzyme involved in the biosynthesis of glycoglycosaminoglycans (GAGs) in tendon tissue.

29. A method of identifying a compound useful in the treatment of tendinopathy comprising administering a test compound to a subject in need of treatment and measuring the ability of the compound to inhibit the activity of an enzyme involved in the synthesis of glycoglycosaminoglycans in tendon tissue.

30. The method according to claim 29 further comprising the steps of

(a) providing at least one sample component selected from the group consisting of CS-GalNAcT-1 , CS-GalNAct-2, heparin sulfate (glucosamine) 3-O-sulfotransferase 1 , GalNT-1 , and Sox9;

(b) combining the sample with a test compound;

(c) measuring the activity of the sample component in response to the test compound; and

(d) determining whether the test compound inhibits the activity of the sample component.

31. Use of a compound that reduces the formation of cartilage-specific proteoglycans in the manufacture of a medicament for treating tendinopathy.

32. The use according to claim 31 , wherein the compound reduces the activity of an enzyme or other protein involved in the synthesis of cartilage.

33. The use according to claim 32, wherein the compound directly or indirectly inhibits the function of the enzyme or protein.

34. The use according to claim 32 or 33, wherein the compound inhibits expression of the enzyme oι* protein.

35. The use according to any one of claims 32 to 34, wherein the enzyme is selected from the group consisting of UDP-D-xylose:core protein β-D- xylosyltransferases, GaI transferases, GIcA transferases, GIcNAc transferases, sulfotransferases, chondroitin sulfate synthases, and heparin sulfate sulfotransferases.

36. The use according to claim 35, wherein the enzyme is selected from the group consisting of chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CS-GaI NAcT-1), chondroitin sulfate N-acetylgalactosaminyltransferase 2 (CS- GalNAcT-2), galactosamine polypeptide N-acetylgalactosaminyltransferase 1 (GalNAcT-1), and heparin sulfate (glucosamine) 3-O-sulfotransferase 1 (Hs3st1).

37. The use according to claim 31 , wherein the compound reduces expression of a cartilage-specific structural protein.

38. The use according to claim 31 , wherein the compound reduces the activity of transcription factors involved in the expression of the cartilage-specific structural protein.

39. The use according to claim 38, wherein the transcription factor is Sox9.

40. The use according to any one of claims 31 to 39, wherein the proteoglycan is selected from the group consisting of aggrecan, versican, syndecan-3, and type Il collagen.

41. The use according to any one of claims 31 to 40, wherein the tendinopathy is selected from the group consisting of tendonitis, tendinitis, tendonosis, paratendinitis, tenosynovitis, tendon injury, tendon trauma, perintendinitis, and paratenonitis.

42. The use according to any one of claims 31 to 41 , wherein the medicament is administered to a patient is selected from the group consisting of primates, monkeys, rodents, sheep, rabbits, dogs, guinea pigs, horses, cows, and cats.