Method and means for treatment of osteoarthritis

Abstract

The present invention relates to in vivo and in vitro methods, agents and compound screening assays for inducing anabolic stimulation of chondrocytes, including cartilage formation enhancing pharmaceutical compositions, and the use thereof in treating and/or preventing a disease involving a systemic or local decrease in mean cartilage thickness in a subject.

1 - 32 . (canceled) 
     
     
         33 . A method for identifying a compound that induces chondrocyte anabolic stimulation, comprising (a) contacting a compound with a polypeptide comprising an amino acid sequence of SEQ ID NO: 480, in an in vitro cell-free preparation; (b) measuring the binding affinity of said compound to said polypeptide; and (c) selecting a compound for confirmation as an inducer of chondrocyte anabolic stimulation, which compound is selected based on its binding affinity for the polypeptide of SEQ ID NO. 480. 
     
     
         34 . A method according to  claim 33  for identifying a compound that induces chondrocyte anabolic stimulation in a mammalian cell, said method further comprising
 (d) contacting said compound selected to have binding affinity to, and capable of forming a complex with, said polypeptide of SEQ ID NO: 480 with a mammalian cell, which expresses a protein that is a normal constituent of cartilage and/or that is required for the formation of cartilage, which is in culture, and in which said polypeptide comprising the amino acid sequence of SEQ ID NO: 480 is expressed; and   (e) measuring in said culture levels of at least protein that is a normal constituent of cartilage and/or that is required for the formation of cartilage; and   (f) determining if said levels of said protein which is a normal constituent of cartilage and/or which is required for the formation of cartilage, are increased as compared to levels of said one or more protein in said mammalian cell that is not contacted with said compound; and   (f) selecting a compound, based on its increase in level of said protein, for confirmation as an inducer of chondrocyte anabolic stimulation in a mammalian cell.   
     
     
         35 . A method according to  claim 34 , wherein said one or more protein that is a normal constituent of cartilage and/or that is required for the formation of cartilage is a protein selected from the group consisting of collagen type II, alpha-1 (col2α1) and aggrecan. 
     
     
         36 . The method according to  claim 34  wherein said compound having binding affinity to, and capable of forming a complex with, said polypeptide of SEQ ID NO: 480 exhibits a binding affinity of at least 10 micromolar. 
     
     
         37 . The method according to  claim 34 , wherein said mammalian cell are a mixture of chondrocytes and de-differentiated chondrocytes. 
     
     
         38 . An agent for inducing the anabolic stimulation of chondrocytes, wherein said agent consists of (1) a targeting nucleic acid sequence consisting of a sequence that is complementary to at least about 17 nucleotides of the polyribonucleotide sequence of SEQ ID NO: 479, and (2) one or more other nucleic acid sequences that are not complementary to said nucleic acid sequence. 
     
     
         39 . A vector comprising the agent according to  claim 38 , wherein said vector is capable of expressing said agent in a mammalian cell. 
     
     
         40 . The vector according to  claim 38 , which is an adenoviral, retroviral, adeno-associated viral, lentiviral, a herpes simplex viral or a sendaiviral vector. 
     
     
         41 . The agent according to  claim 38 , wherein a first sequence of said other nucleic acid sequences that are not complementary to said nucleic acid sequence in said undifferentiated mammalian cells is complementary to said targeting nucleotide sequence. 
     
     
         42 . The agent according to  claim 41 , wherein a second sequence of said other nucleic acid sequences that are not complementary to said nucleic acid sequence in said undifferentiated mammalian cells consists of a loop region connecting said targeting nucleotide sequence and said first sequence of other nucleic acid sequences complementary to said targeting nucleic acid sequence. 
     
     
         43 . The agent according to  claim 42 , wherein said loop region comprises a nucleic acid sequence of SEQ ID NO: 83. 
     
     
         44 . A chondrocyte anabolic stimulation enhancing pharmaceutical composition comprising a therapeutically effective amount of an agent of  claim 41  in admixture with a pharmaceutically acceptable carrier. 
     
     
         45 . A method of treating and/or preventing a disease involving a systemic or local decrease in cartilage in a subject suffering from or susceptible to the disease, comprising administering to said subject a pharmaceutical composition according to  claim 44 . 
     
     
         46 . A method according to  claim 45 , wherein said agent is administered locally to the site of treatment of said patient. 
     
     
         47 . The method according to  claim 45  wherein the disease is selected from the group consisting of osteoarthritis, rheumatoid arthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, algodystrophy, Tietze syndrome or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, osteoarthritis deformans endemica, Mseleni disease, Handigodu disease, degeneration resulting from fibromyalgia, systemic lupus erythematosus, scleroderma, ankylosing spondylitis, hereditary chondrolysis, chondrodysplasias, pseudoachondrodysplasias, microtia, anotia, and metaphyseal chondrodysplasia. 
     
     
         48 . The method according to  claim 47 , wherein the disease is osteoarthritis. 
     
     
         49 . A method for in vitro production of cartilage tissue, comprising the steps of contacting de-differentiated chondrocyte cells with an effective amount of agent according to  claim 41 , for a time sufficient to re-differentiate the chondrocytes, thereby producing a cartilaginous matrix. 
     
     
         50 . The method according to  claim 49 , comprising the steps of:
 applying a mixture of chondrocytes and de-differentiated chondrocytes on a substrate to form a cellular substrate, and   contacting said cells with an effective amount of said agent, thereby producing a continuous cartilaginous matrix.   
     
     
         51 . A method for diagnosing a pathological condition involving a systemic or local decrease in mean cartilage thickness or a susceptibility to the condition in a subject, comprising determining the amount of polypeptide comprising an amino acid sequence of SEQ ID NO: 480 in a biological sample obtained from said subject, and comparing said first amount with the ranges of amounts of the polypeptide determined in a population of healthy subjects, wherein an increase of the amount of polypeptide in said biological sample compared to the range of amounts determined for healthy subjects is indicative of the presence of the pathological condition. 
     
     
         52 . A method for diagnosing a pathological condition involving chondrocyte de-differentiation, said method comprising the steps of:
 determining the nucleic acid sequence of the gene coding for the protein of SEQ. ID NO. 480 in a genomic DNA sample;   comparing the sequence from step (a) with the nucleic acid sequence of a healthy subject; and   identifying any difference(s) related to the pathological condition.   
     
     
         53 . Method according to  claim 51 , wherein the pathological condition is osteoarthritis.