Wu X, Holschen J, Kennedy S C, Ponder K P
Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
Hum Gene Ther. 1996 Jan 20;7(2):159-71. doi: 10.1089/hum.1996.7.2-159.
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 alpha 1-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.
尽管逆转录病毒载体在基因治疗方面显示出前景，但它们在动物体内的表达水平一直较低。更好地理解逆转录病毒载体中启动子的功能应有助于设计出改进的载体。在本研究中，将肝脏特异性启动子克隆到逆转录病毒载体中，并分析了来自逆转录病毒长末端重复序列（LTR）和内部启动子的表达。此外，将寡聚化的肝脏特异性转录因子结合位点置于每个启动子的上游，试图进一步提高表达。额外的寡聚化结合位点仅略微增加了表达或在肝癌细胞中抑制了表达，这表明这不是增加逆转录病毒载体表达的有效方法。出乎意料的是，肝脏特异性白蛋白启动子在成纤维细胞中从逆转录病毒载体高水平表达，这表明逆转录病毒元件起到了增强子的作用。此外，在白蛋白启动子附近添加肝细胞核因子4（HNF-4）结合位点抑制了成纤维细胞中的LTR和白蛋白启动子，这种效应可能是由非肝细胞中存在的可与HNF-4位点结合的抑制蛋白介导的。这些结果表明，正向和负向影响都可以在LTR和白蛋白启动子之间传递。相比之下，肝脏特异性人α1-抗胰蛋白酶启动子似乎不符合这两个标准中的任何一个与LTR相互作用。逆转录病毒载体具有可能在体内抑制LTR和一些内部启动子表达的序列。我们假设在组织培养中不与LTR相互作用的内部启动子在体内将对逆转录病毒序列的抑制作用具有抗性。
