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 . Method for identifying a compound that induces chondrocyte anabolic stimulation, comprising 
 contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82 and 198-391; and    measuring a compound-polypeptide property related to the anabolic stimulation of chondrocytes.    
     
     
         2 . The method according to  claim 1 , wherein said polypeptide is in an in vitro cell-free preparation.  
     
     
         3 . The method according to  claim 2 , wherein said polypeptide is present in a mammalian cell.  
     
     
         4 . The method of  claim 1 , wherein said property is a binding affinity of said compound to said polypeptide.  
     
     
         5 . The method of  claim 3 , wherein said property is activation of a biological pathway producing a biochemical marker indicative of the anabolic stimulation of chondrocytes.  
     
     
         6 . The method of  claim 5  wherein said indicator is selected from the group consisting of collagen type II, alpha-1 (col2α1) and aggrecan.  
     
     
         7 . The method of  claim 6  wherein said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82.  
     
     
         8 . The method according to  claim 1 , wherein said compound is selected from the group consisting of compounds of a commercially available screening library and compounds having binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82 and 198-391.  
     
     
         9 . The method according to  claim 2 , wherein said compound is a peptide in a phage display library or an antibody fragment library.  
     
     
         10 . An agent for inducing the anabolic stimulation of chondrocytes, selected from the group consisting of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally-occurring polynucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82 and 198-391.  
     
     
         11 . The agent according to  claim 10 , wherein polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82.  
     
     
         12 . The agent according to  claim 11 , wherein a vector in a mammalian cell expresses said agent.  
     
     
         13 . The agent according to  claim 12 , wherein said vector is an adenoviral, retroviral, adeno-associated viral, lentiviral, a herpes simplex viral or a sendaiviral vector.  
     
     
         14 . The agent according to  claim 10 , wherein said antisense polynucleotide and said siRNA comprise an antisense strand of 17-25 nucleotides complementary to a sense strand, wherein said sense strand is selected from 17-25 continuous nucleotides of a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82.  
     
     
         15 . The agent according to claims  14 , wherein said siRNA further comprises said sense strand.  
     
     
         16 . The agent according to  claim 15 , wherein said sense strand is selected from 17-25 continuous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-28.  
     
     
         17 . The agent according to  claim 16 , wherein said siRNA further comprises a loop region connecting said sense and said antisense strand.  
     
     
         18 . The agent according to  claim 17 , wherein said loop region comprises a nucleic acid sequence defined of SEQ ID NO: 83.  
     
     
         19 . The agent according to  claim 18 , wherein said agent is an antisense polynucleotide, ribozyme, or siRNA comprising a nucleic acid sequence complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 84-197.  
     
     
         20 . A chondrocyte anabolic stimulation enhancing pharmaceutical composition comprising a therapeutically effective amount of an agent of  claim 10  in admixture with a pharmaceutically acceptable carrier.  
     
     
         21 . A method of treating and/or preventing a disease involving a systemic or local decrease in cartilege in a subject suffering from or susceptible to the disease, comprising administering to said subject a pharmaceutical composition according to  claim 20 .  
     
     
         22 . The method according to  claim 21  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.  
     
     
         23 . The method according to  claim 22 , wherein the disease is osteoarthritis.  
     
     
         24 . Use of an agent according to claims  10 - 19  in the manufacture of a medicament for the treatment and/or prevention of a disease involving a decrease in mean cartilage thickness.  
     
     
         25 . Use according to  claim 24 , wherein the disease is selected from the group consisting of osteoartnritis, hypercalcemia of malignancy, multiple myelomatosis, hyperparathyroidism, and hyperthyroidism.  
     
     
         26 . Use according to  claim 25 , wherein the disease is osteoarthritis.  
     
     
         27 . A method for in vitro production of cartilage tissue, comprising contacting de-differentiated chondrocyte cells with a polynucleotide sequence comprising a nucleic acid sequence complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 1-28 for a time sufficient to re-differentiate the chondrocytes, thereby producing a cartilaginous matrix.  
     
     
         28 . A method according to  claim 27 , comprising: 
 applying a mixture of chondrocytes and de-differentiated chondrocytes on a substrate to form a cellular substrate, and    contacting said cells with a polynucleotide sequence comprising a nucleic acid sequence complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 84-197, thereby producing a continuous cartilaginous matrix.    
     
     
         29 . 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 a first amount of polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82 and 198-391 present 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.  
     
     
         30 . A method for diagnosing a pathological condition involving chondrocyte de-differentiation, said method comprising the steps of: 
 determining the nucleic acid sequence of any one of the genes of SEQ. ID NOS. 1-28 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.    
     
     
         31 . A method for diagnosing a pathological condition involving chondrocyte de-differentiation, said method comprising the steps of: 
 determining the amount of any one of the polypeptides of SEQ ID NOS. 55-82 in a sample; and    comparing the amount determined in step (a) with the amount of said polypeptide in a sample from a healthy individual;    wherein an increase as compared to the sample of the healthy individual is indicative for the onset or presence of said pathological condition.    
     
     
         32 . Method according to  claim 31 , wherein the pathological condition is osteoarthritis.