Tnf-α ribozymes and antisense molecules as potential therapeutic agents for treatment of rheumatoid arthritis

Rheu matoid arthritis(RA) is an autoimmune disease of unknown etiology. Inflammatory cytokines such as tumor necrosis factor alpha(TNF-a) are key factors which drive the inflammatory response leading to joint destruction in RA. Controlling the production or expression of this damaging cytokine holds future promise for therapeutic intervention. The objective of this study is to develop TNF-a ribozyme and antisense molecules to "knock-out" this disease related cytokine in cultures of RA synovial fibroblasts, RA synovium and the mouse model of arthritis. TNF-a ribozyme and antisense molecules were cloned into pcDNA3-Neo and transfected into cultures of RA synovial fibroblasts which produce constitutive levels of TNF-a, collagenase and stromelysin mRNAs . Our results show that the 'FNF-a ribozyme and antisense molecules suppress TNF-a mRNA levels by 65%and 28%, respectively. ]'he "downstream" effects of TNF-a "knock-out" on the expression of collagenase and stromelysin mRNAs were also measured. The TNF-a ribozyme and antiseuse molecules suppressed stromelysin mRNA levels by 89% and 46 .respeclively, relative to the control vector. The TNF-a ribozyme and antisense molecules also suppressed collagenase mRNA levels by 48% and 35% .respectively. relative to the control vector. Overall, the TNF-a ribozyme appeared to he more effective at reducing mRNA levels for all three species of mRNAs than the TNF-a antisense construct.