Robinson D, Elliott J F, Chang L J
Department of Medical Microbiology and Infectious Diseases, University of Alberta, Edmonton, Canada.
Gene Ther. 1995 Jun;2(4):269-78.
We have constructed a new retroviral vector by making modifications to the commonly used Moloney murine leukemia virus (MoMLV) based vector in the long terminal repeat (LTR). The changes include replacement of a portion of the U3 region of the MoMLV LTR with a hybrid regulatory element consisting of the human cytomegalovirus immediate-early enhancer/promoter (CMV-IE) together with the human immunodeficiency virus transactivation response element (HIV-TAR). Transfection of chloramphenicol acetyl transferase (CAT) reporter constructs into a variety of human cell lines showed that the hybrid LTR with the CMV-IE/HIV-TAR enhancer/promoter exhibited basal expression levels which were 10- to 50-fold higher than those obtained from the wild-type MoMLV-LTR enhancer/promoter. Expression from the recombinant LTR was further increased in the presence of the HIV-Tat protein, and surprisingly, Tat up-regulated transcription from both the HIV and the MoMLV TATA boxes. In contrast, a MoMLV enhancer/promoter containing only the HIV-TAR element in the LTR did not respond to Tat. When stably transfected into an amphotropic packaging cell line, the modified retroviral vector containing the hybrid LTR plus an extended packaging signal consistently gave higher titres of retrovirus than did the parental MoMLV based vector. Higher basal expression levels which can be further upregulated by Tat, together with more efficient virion production, suggests that the modified vector should be superior for anti-HIV gene therapy applications as well as for other more general applications in human gene therapy.
我们通过对常用的基于莫洛尼鼠白血病病毒（MoMLV）的载体的长末端重复序列（LTR）进行改造，构建了一种新的逆转录病毒载体。这些改变包括用一个由人巨细胞病毒立即早期增强子/启动子（CMV-IE）和人免疫缺陷病毒反式激活应答元件（HIV-TAR）组成的混合调控元件替换MoMLV LTR的U3区域的一部分。将氯霉素乙酰转移酶（CAT）报告构建体转染到多种人类细胞系中，结果显示，带有CMV-IE/HIV-TAR增强子/启动子的混合LTR的基础表达水平比野生型MoMLV-LTR增强子/启动子高10至50倍。在HIV-Tat蛋白存在的情况下，重组LTR的表达进一步增加，而且令人惊讶的是，Tat上调了来自HIV和MoMLV TATA框的转录。相比之下，LTR中仅含HIV-TAR元件的MoMLV增强子/启动子对Tat没有反应。当稳定转染到嗜性包装细胞系中时，含有混合LTR加延伸包装信号的改造逆转录病毒载体产生的逆转录病毒滴度始终高于亲本的基于MoMLV的载体。更高的基础表达水平可被Tat进一步上调，以及更有效的病毒粒子产生，这表明改造后的载体在抗HIV基因治疗应用以及人类基因治疗的其他更普遍应用中应该更具优势。
