Ribozymes as tools for therapeutic target validation in arthritis

In this paper me describe a method for validating therapeutic gene targets in arthritic disease. Ribozymes are catalytic oligonucleotides capable of h ighly sequence-specific cleavage of RNA. We designed ribozymes that cleave the mRNA encoding stromelysin, a matrix metalloproteinase implicated in car tilage catabolism. Ribozymes were initially screened in cultured fibroblast s to identify sites in the mRNA that were accessible for binding and cleava ge. Accessible sites for ribozyme binding were found in various regions of the mRNA, including the 5' untranslated region, the coding region, and the 3' untranslated region, Several ribozymes that mediated sequence-specific a nd dose-dependent inhibition of stromelysin expression were characterized. Site selection in cell culture was predictive of in vivo bioactivity. An as say for measuring cartilage catabolism in rabbit articular cartilage explan ts was developed. Ribozymes inhibited IL-1-stimulated stromelysin mRNA expr ession in articular cartilage explants, yet failed to inhibit proteoglycan degradation. This indicated that up-regulation of stromelysin was not essen tial for IL-1-induced cartilage catabolism, Broad applications of this appr oach in therapeutic target validation are discussed.