RETROVIRAL VECTOR SEQUENCES MAY INTERACT WITH SOME INTERNAL PROMOTERSAND INFLUENCE EXPRESSION

Although retroviral vectors show promise for gene therapy, their expre ssion in animals has been low. An improved understanding of how promot ers function from a retroviral vector should facilitate the design of improved vectors. In this study, liver-specific promoters were cloned into a retroviral vector and expression from the retroviral long termi nal repeat (LTR) and the internal promoter was analyzed. In addition, oligomerized liver-specific transcription factor binding sites were pl aced upstream of each promoter in an attempt to increase expression fu rther. Additional oligomerized binding sites only increased expression slightly or inhibited expression in hepatoma cells, suggesting that t his is not an effective way to increase expression from a retroviral v ector. Unexpectedly, the liver-specific albumin promoter was expressed at high levels from a retroviral vector in fibroblasts, suggesting th at retroviral elements functioned as an enhancer. Furthermore, the add ition of HNF-4 binding sites adjacent to the albumin promoter inhibite d both the LTR and albumin promoter in fibroblasts, an effect that was probably mediated by inhibitory proteins present in nonhepatic cells that can bind to HNF-4 sites. These results suggest that both positive and negative influences can be transmitted between the LTR and the al bumin promoter. In contrast, the liver-specific human alpha(1)-antitry psin promoter did not appear to interact with the LTR by either of the se criteria. Retroviral vectors have sequences that may inhibit expres sion of the LTR and some internal promoters in vivo. We hypothesize th at internal promoters that do not interact with the LTR in tissue cult ure will be resistant to inhibitory effects of retroviral sequences in vivo.