Evaluation of safety and efficacy of anti-HIV-1 shRNA expression in vivo and vitro

Double-stranded RNA expressed as short hairpin RNA (shRNA) can induce gene silencing through a process known as RNA interference (RNAi). Previously, we have shown that stable expression of anti-HIV-1 shRNAs with lentiviral vec- tors results in strong inhibition of virus replication. However, the virus evolves to become RNAi resistant. Targeting highly conserved regions restricts the viral escape possibilities and the virus mutants that are selected closely resemble natural variation in HIV-1 sequences. Viral escape can be prevented by the use of a combination of shRNAs. For the clinical application of RNAi, transduction of hematopoietic stem cells is desirable, which will generate a constant supply of HIV-resistant cells. To test the feasibility of such an approach, we used CD34+ precursor cells with a lentiviral vector encoding an anti-HIV-1 shRNA targeting the Nef region. These cells were engrafted in Rag2(-/-)gamma(c)(-/-) mice that allow development of a human immune system. Development and maturation of the human immune system was not affected in the shNef-engrafted mice (cell numbers, cell types). After establishment of the immune system in vivo, we challenged the human CD4+ T cells from these mice with HIV-1 ex vivo. Potent sequence specific inhibition of HIV-1 was scored. In addition, in HIV-1 infected cell cultures we measured preferential outgrowth of the shRNA-expressing cells over untreated cells, a promising result for our gene therapy approach. Our latest results from in vivo and in vitro experiments in which we evaluate safety and efficacy will be presented.