678. Inflammatory Cytokines Regulate Transgene Expression from Rheumatoid Synoviocytes Infected with Double Stranded Adeno-Associated Virus Vectors

Abstract

A major drawback to current gene transfer strategies is the inability to regulate transgene expression. An ideal vector would provide local transgene expression at the site of the disease only when the disease is active. Recombinant adeno-associated virus (rAAV) appears to possess these important qualities. We monitored the effects of inflammatory cytokines on transgene expression in human rheumatoid arthritis fibroblast-like synoviocytes (FLS) infected with rAAV. Synovial cell cultures were infected with rAAV under control of the cytomegalovirus (CMV) promoter in the presence or absence of TNF|[alpha]|, IL-1|[beta]|, and/or IL-6. These cytokines were found to act synergistically to increase transgene expression in rAAV infected FLS up to 1000 fold. The enhanced expression required the continued presence of cytokines, although cytokine removal and re-addition demonstrated a remarkable ability of the cytokines to regulate transgene expression. The effect was most pronounced on cells infected with a double stranded version of AAV in which a rate-limiting step of viral transduction (second strand synthesis) is bypassed, and the effect was not promoter specific, as similar results were obtained using rAAV under control of the chicken beta actin promoter. mRNA levels of the transgene were demonstrated to increase up to 25-fold upon cytokine addition. The observed phenomenon was also phosphatidylinositol 3-kinase (PI3K) dependent, as addition of wortmannin reversibly inhibited cytokine-mediated increases in transgene expression. These results suggest that expression of a therapeutic transgene can be controlled by the presence of inflammation following AAV-mediated gene transfer, making AAV an attractive gene transfer vector for chronic inflammatory diseases such as rheumatoid arthritis.