Retroviral vector with a CMV-IE/HIV-TAR hybrid LTR gives high basal expression levels and is up-regulated by HIV-1...

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.