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High-Affinity Rev-Binding RNA Molecules Selected In Vitro are Effective Inhibitors of HIV-1 Gene Expression and Replication L. KOFELDT*1, B. WILLIAMSON1, V. JOHNSON1, A. SMITH1, L. GIVER2, T. SYMENSMA2, A. ELLINGTON2, and D. CAMERINI1. 1Univ. of Virginia, Charlottesville; and 2Univ. of Texas, Austin Small RNA molecules containing the core Rev binding element (RBE) have been shown to inhibit HIV-1 replication and Rev-dependent gene expression. Two RNA molecules that bind Rev with higher affinity than the wild-type RBE (RBE aptamers) were derived from random sequence libraries by an in vitro selection process known as SELEX. These RBE aptamers were shown to substitute functionally for the RBE in assays of Rev-dependent gene expression using a CAT reporter gene. We have tested their ability to inhibit Rev-dependent HIV-1 gene expression in two tissue culture assay systems. The RBE aptamers and wild-type RBE were fused to a human tRNAmet gene with the tRNAmet gene alone used as a control. These constructs were co-transfected with a Rev expression vector and a Rev-dependent HIV-1 gag-pol expression vector into COS cells. Both aptamers and wild-type RBE inhibited production of p24 by 85–95% compared to the control. CEM cells were transduced with the aptarner constructs and a marker gene and subsequently were infected with a molecular clone of HIV-1. One RBE aptamer and wild-type RBE decreased the number of cells expressing p24 by >50%. Virus production was decreased 2–7 fold in these cell lines as compared to the control. CD34+ cells derived from human fetal liver were transduced with the aptamer constructs or wild-type RBE as well as a marker gene and were injected into the human thymus/liver grafts of sublethally irradiated SCID-hu mice. The marker gene was expressed by 25–95% of the thymocytes eight weeks post gene transfer. Key Words: Aptamer, Gene Therapy, REV Decoy |
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© 7th
Conference on Retroviruses and Opportunistic Infections, |