Retroviral Vector Sequences May Interact with Some Internal Promoters and Influence Expression

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Abstract
Although retroviral vectors show promise for gene therapy, their expression in animals has been low. An improved understanding of how promoters 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 terminal repeat (LTR) and the internal promoter was analyzed. In addition, oligomerized liver-specific transcription factor binding sites were placed upstream of each promoter in an attempt to increase expression further. Additional oligomerized binding sites only increased expression slightly or inhibited expression in hepatoma cells, suggesting that this is not an effective way to increase expression from a retroviral vector. Unexpectedly, the liver-specific albumin promoter was expressed at high levels from a retroviral vector in fibroblasts, suggesting that retroviral elements functioned as an enhancer. Furthermore, the addition of HNF-4 binding sites adjacent to the albumin promoter inhibited 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 albumin promoter. In contrast, the liver-specific human α1-antitrypsin promoter did not appear to interact with the LTR by either of these criteria. Retroviral vectors have sequences that may inhibit expression of the LTR and some internal promoters in vivo. We hypothesize that internal promoters that do not interact with the LTR in tissue culture will be resistant to inhibitory effects of retroviral sequences in vivo. Although retroviral vectors can result in long-term expression in hepatocytes of animals, they are poorly expressed in vivo. An understanding of how an internal promoter functions from a retroviral vector should lead to the development of improved vectors. The results presented here provide evidence that some promoters such as the albumin promoter can interact with retroviral elements. This interaction results in the activation of the liver-specific albumin promoter in fibroblast-derived cells and the transmission of a negative influence located just upstream of the albumin promoter to the LTR promoter. In contrast, other promoters such as the α1-antitrypsin promoter do not appear to interact with retroviral sequences. There are several elements in and adjacent to the LTR that inhibit expression in some cells. We hypothesize that an inhibitory effect will only be transmitted to internal promoters which can interact with the LTR.