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Ribozymes as Tools for Therapeutic Target Validation in Arthritis

Thale C. Jarvis^1,, Karyn S. Bouhana^*, Mark E. Lesch, Suzy A. Brown, Tom J. Parry^*, Denis J. Schrier, Stephen W. Hunt, III, Pamela A. Pavco^* and Craig M. Flory

^* Ribozyme Pharmaceuticals, Boulder, CO 80301; Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105; and Atugen USA, Boulder, CO 80301

In this paper we describe a method for validating therapeutic gene targets in arthritic disease. Ribozymes are catalytic oligonucleotides capable of highly sequence-specific cleavage of RNA. We designed ribozymes that cleave the mRNA encoding stromelysin, a matrix metalloproteinase implicated in cartilage catabolism. Ribozymes were initially screened in cultured fibroblasts to identify sites in the mRNA that were accessible for binding and cleavage. 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 and dose-dependent inhibition of stromelysin expression were characterized. Site selection in cell culture was predictive of in vivo bioactivity. An assay for measuring cartilage catabolism in rabbit articular cartilage explants was developed. Ribozymes inhibited IL-1-stimulated stromelysin mRNA expression in articular cartilage explants, yet failed to inhibit proteoglycan degradation. This indicated that up-regulation of stromelysin was not essential for IL-1-induced cartilage catabolism. Broad applications of this approach in therapeutic target validation are discussed.

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