810. Comparison of Anti-HIV Activity from Various Lentiviral Vectors Carrying Anti-HIV shRNA In Vitro
Authors: Shirley Li, Haitang Li, Daniela Castanotto, Keith Meinking, Guihua Sun, John A. Zaia, John J. Rossi
Journal: Molecular Therapy (2006)

Eight pHIV-7-GFP lentiviral vectors encoded anti-HIV-1 small hairpin RNA (shRNA) were constructed. Three target sites in the HIV genome were selected to construct the shRNAs. The first site (SI) resides in a region of rev that overlaps with the adjacent tat gene, while the second (SII) and third sites (SIII) are selected in conserved regions that reside exclusively in rev or tat. The expression and functionality were compared using snRNA Pol III U6 versus Pol II U1 promoters in the following constructs: U1-S1: shRNA targets SI controlled by a U1 promoterU1-S2: shRNA targets SII controlled by a U1 promoterU6-S2: shRNA targets SII controlled by a U6 promoterU1-3S: Three shRNAs individually target SI, SII and SIII controlled by one U1 promoterU6-3S: Three shRNAs individually target SI, SII and SIII controlled by one U6 promoter driving from 5' to 3'U6-3SF: Three shRNAs individually target SI, SII and SIII controlled by one U6 promoter driving from 3' to 5'3U6-3S: Three shRNAs individually target SI, SII and SIII controlled by separate U6 promoters for each shRNAU1-S2-U6-S2: Two shRNAs target SII controlled by either U1 or U6 promotersControl: Lentiviral vector encoding GFP only All vectors were transducted into CEM cells, sorted for eGFP positive cells, challenged with HIV-1 strains IIIB and NL4-3, observed for HIV RT activities in culture supernatant, and evaluated for anti-HIV activity by comparison with control. All constructs demonstrated inhibition of HIV-1, but U1-S1 showed the most potent HIV-1 inhibition of both strains of virus with 2-3 log reduction of supernatant RT activity. In comparison, the other constructs produced 1-2 logs of inhibition. The comparison of U1-S2 vs U6-S2; three shRNAs controlled by either U1 or the U6 promoters (U1-3S vs U6-3S) and the single or multiple shRNAs controlled by the same promoter (U1-S2 vs U1-3S or U1-S2-U6-S2; and U6-S2 vs U6-3S or U6-3SF or 3U6-3S) all showed no further significant differences in inhibition. Subsequent investigation attributed this to lack of proper processing of the hairpins from the single promoter, whereas the three independent promoters showed toxicity when challenged with HIV-1. The three independent promoters were shown to be producing large amounts of the shRNAs, perhaps contributing to the toxicity. Thus, with shRNAs, an increase in RNAi does not necessarily contribute to an improved antiviral effect. Current studies are investigating the differences in the Pol III and Pol II expression systems that might affect the efficacy of the shRNAs, including the possibility that the U1-transcribed shRNAs use different export pathways alleviating the competition for export with the endogenous microRNAs. Finally, these results demonstrate that the anti-HIV-1 tat and rev specific shRNAs expressed from U1 pol II promoter in lentiviral vectors provide an important potential advantage for AIDS gene therapy and will be used in primary cells for preclinical study necessary for further clinical application.