HIV-1 LTR as a target for synthetic ribozyme-mediated inhibition of gene expression: site selection and inhibition in cell culture

A library of three synthetic ribozymes with randomized arms, targeting NUX, GUX and NXG triplets, respectively, were used to identify ribozyme-accessi ble sites on the HIV-1 LTR transcript comprising positions -533 to 385. Thr ee cleavable sites were identified at positions 109, 115 and 161. Ribozymes were designed against these sites, either unmodified or with 2'-modificati ons and phosphorothioate groups, and their cleavage activities of the trans cript were determined. Their biological activities were assessed in cell cu lture, using a HIV-1 model assay system where the LTR is a promoter for the expression of the reporter gene luciferase in a transient expression syste m. Intracellular efficiency of the ribozymes were determined by cotransfect ion of ribozyme and plasmid DNA, expressing the target RNA. Modified ribozy mes, directed against positions 115 and 161, lowered the level of LTR mRNA in the cell resulting in inhibition of expression of the LTR-driven reporte r gene luciferase of 87 and 61%, respectively. In the presence of Tat the i nhibitions were 43 and 25%. The inactive variants of these ribozymes exhibi ted a similar inhibitory effect. RNase protection revealed a reduction of R NA which was somewhat stronger for the active than the inactive ribozymes, particularly for ribozyme 115. Unmodified ribozymes showed no inhibition in the cell. The third ribozyme, targeting a GUG-triplet at position 109, pos sessed only low cleavage activity in vitro and no inhibitory effect in cell culture.