369. Selective Expansion of Human T Lymphocytes Transduced by HIV Vectors Containing IMPDH2 and Anti-HIV siRNA Genes

Abstract

To achieve effective protection of T cells from HIV infection using gene therapy approaches, a sufficient number of T cells expressing anti-HIV gene(s) are required. However, vectors derived from Moloney murine leukemia virus or human immunodeficiency virus (HIV) are relatively inefficient at delivering foreign genes into hematopoietic stem cells (HSCs) from which the genetically modified T cells could be derived. In this study, we evaluated a mutated human type II inosine-5′ monophosphate dehydrogenase (IMPDH2) for its ability to confer cell resistance to drugs such as mycophenolic acid (MPA) and its precursor mycophenolate mofetil (MMF) (Serrano et al. Mol Therapy 3:S152, 2001). The safety profile of these drugs in clinics may allow direct selection and expansion of the transduced T cell in vivo. The cDNA for the mutant IMPDH2, with two amino acid substitutions, was placed under the control of the promoter of Rous sarcoma virus (RSV) and inserted into pHIV7, a 3rd generation HIV vector containing the GFP gene. H9 cells were transduced with the IMPDH2 vector and selected in the following escalating doses of MPA: 3, 6, 12, 18 and 24 × 10−6 M for 24 days. The viability at these doses was 94, 94, 71, 51 and 12%, respectively, whereas cells transduced with a control GFP vector were eliminated at all doses. The percentage of GFP increased from 5.2% before drug selection to 90–97% after drug selection, suggesting that IMPDH2 could serve as an effective selection marker for the transduced cells. To treat HIV infection, we evaluated a small interfering RNA (siRNA) specific against the p24 coding region for its ability to down-regulate HIV replication. A hairpin p24 siRNA gene was placed under the control of the H1 promoter and inserted into pHIV7. H9 cells were transduced with this vector and challenged with NL4-3 at different doses. Our results showed an effective inhibition of HIV replication, from 2 to 6 log reduction, by the p24 siRNA for up to 30 days after challenge in culture. The IMPDH2 cDNA was then inserted into the vector containing the siRNA gene and the effect of drug selection on protection of the transduced cells from HIV infection is under investigation.