Inhibition of Human Immunodeficiency Virus Type-1 by Retroviral Vectors Expressing Antisense-TAR

Abstract
The human immunodeficiency virus type-1 (HIV-1) Tat activation response (TAR) region is essential for Tat-mediated trans-activation of the HIV-1 long terminal repeat (LTR). The TAR element is present on the 5′ and 3′ ends of all HIV-1 transcripts and is relatively conserved among different HIV-1 isolates. These properties make it an attractive target for anti-HIV-1 gene therapy strategies. We have constructed a Moloney murine leukemia-based retroviral vector that expresses a chimeric tRNAiMet-antisense TAR fusion transcript complementary to the HIV-1 TAR region. The potential of this anti-TAR retroviral vector to inhibit HIV-1 was initially tested by transient transfections with an HIV-1–LTR–Tat expression plasmid into HeLa–CAT cells. Anti-TAR inhibited Tat-mediated HIV-1 LTR-driven CAT reporter gene expression in a dose-dependent fashion. The antisense-TAR vector was then used to transduce the human SupT1 T cell line. Cotransfection of these SupT1 cells with a Tat expression plasmid plus an HIV-1 LTR–CAT reporter plasmid resulted in decreased CAT gene expression in comparison to control transduced SupT1 cells. The antisense-TAR engineered SupT1 cell line was then challenged with HIV-1MN. HIV-1 viral production was inhibited in SupT1 cells transduced with the antisense-TAR retroviral vector. Greater inhibition of HIV-1 was observed with antisense-TAR as compared to antisense-Tat expressing retroviral vector. These observations suggest that antisense-TAR retroviral vectors are potentially useful for clinical anti-HIV-1 gene therapy. One of the major goals in anti-HIV gene therapy is to develop an effective, antisense-based anti-HIV strategy that specifically targets essential, conserved, and preferably multiple regions on the HIV-1 mRNA. The HIV-1 TAR region meets these criteria because it is essential for Tat trans-activation, is conserved among primary isolates, and is present on both ends of all HIV-1 mRNA and the viral genome itself. A general advantage of antisense-based strategies, compared with trans-dominant viral proteins, is the lack of immunogenicity. Based on these reasons, we developed an antisense-TAR retroviral vector and demonstrate effective inhibition of HIV-1 in vitro. This antisense retroviral vector therefore represents a potential candidate for clinical anti-HIV gene therapy.