REPRESSION OF RETROVIRUS-MEDIATED TRANSGENE EXPRESSION BY INTERFERONS- IMPLICATIONS FOR GENE-THERAPY

Retrovirus-mediated gene transfer is commonly used in gene therapy pro tocols and has the potential to provide long-term expression of the tr ansgene. Although expression of a retrovirus-delivered transgene is sa tisfactory in cultured cells, it has been difficult to achieve consist ent and high-level expression in vivo. In this investigation, we explo red the possibility of modulating transgene expression by host-derived cytokines. Normal human keratinocytes and dermal fibroblasts were tra nsduced with recombinant retroviruses expressing a reporter gene (lacZ ). Treatment of transduced cells with a proinflammatory cytokine, gamm a interferon (IFN-gamma), significantly reduced lacZ expression to les s than 25% of that of nontreated cells. The inhibition was concentrati on dependent (peak at 5 ng/ml) and time dependent (maximal at 16 h for transcript and 24 h for protein); expression remained repressed in th e continued presence of IFN-gamma but returned to normal levels 24 h a fter IFN-gamma withdrawal. The decrease in beta-galactosidase activity appeared to result from decrease in steady-state lacZ mRNA levels. In hibitors of transcription and translation blocked IFN-gamma-induced re pression, suggesting involvement of newly synthesized protein intermed iates. Similar results were obtained by treatment of transduced cells with IFN-alpha but not with other proinflammatory cytokines, including tumor necrosis factor alpha, interleukin-2 (IL-1), IL-4, and granuloc yte colony-stimulating factor. Although the level of lacZ mRNA was red uced by >70% following IFN treatment, the rate of lacZ transcription w as not significantly different from that for nontreated cells. These r esults suggest that IFN-mediated regulation of transgene expression is at a posttranscriptional level. Interestingly, IFN-gamma also suppres sed transgene expression driven by a cellular promoter (involucrin) in serted in an internal position in the retroviral vector. The presence of the overlapping 3' untranslated regions in transcripts initiated fr om the internal promoter and the long terminal repeat is suggestive of a posttranscriptional regulation, likely at the level of RNA stabiliz ation. These results provide direct evidence for modulatory effects of IFNs on retrovirus-mediated transgene expression and suggest that gen e therapy results may be altered by host inflammatory responses.