372. Optimizing Foamy Virus Vectors That Inhibit HIV Infection

Several important features of foamy virus (FV) vectors support their use in HIV gene therapy including: a) FV vectors can transduce human hematopoietic stem cells with a high frequency, b) wild type FV does not cause disease in humans or non-human primates, c) FV vectors are self-inactivating, d) relative to lentivirus vectors, there is no recombination potential with HIV, and e) FV vector production does not share molecular pathways with HIV. Our goal is to identify clinically relevant vectors that block HIV production and limit the development of resistance.We have tested three published anti-HIV strategies using FV vectors: 1) Humanized RevM10 (hRevM10) - a transdominant REV mutant which interferes with viral RNA transport (Mol Ther 2005), 2) M87o - a membrane bound entry inhibitor which efficiently blocks HIV (J Virol 2004), 3) shRNA (Sh1) - a small hairpin RNA that targets a conserved region of tat and rev (Nat Biotech 2002).FV vectors that express these three molecules have been tested for their ability to inhibit HIV in three assays. Using a lentivirus vector cotransfection system, the Sh1 FV plasmid was significantly more effective than the hRevM10 FV plasmid in blocking lentivirus vector formation (300x compared to 4x). The second assay utilizes CEMx174 cells transduced with FV vectors, infected with HIV NL4-3 , and then followed for HIV production. Cells transduced with the M87o FV vector had significant reduction in p24 productionand HIV virus formation (both >100x) and showed a selective advantage during HIV challenge. Cells transduced with Sh1 demonstrated a transient reduction of both p24 and HIV virus formation (10x) for 3-5 days. hRevM10 showed no activity in this assay. In a third assay, primary human CD34+ cells were transduced, sorted, differentiated into monocytes, and then infected with HIV JR-FL . Monocytes transduced with a FV vector expressing Sh1 demonstrated a 10x decrease in p24 production at day 7 and a 250x at later time points, while HIV virus production was decreased relative to control cells by 120x at day 7 and 2000x at later time points. Cells transduced with hRevM10 had a 2x decrease in both p24 and HIV viral production, but lacked statistical significance. The relative efficacy of the three strategies in these three assays is: M87o>Sh1>hRevM10.Based on these encouraging results, we have created combination vectors containing 2 or 3 of these anti-HIV strategies. In the lentivirus cotransfection system, plasmids containing