Combination of anti HIV-1 siRNAs and a fusion inhibitor for genetic treatment of AIDS.

Despite the remarkable success of highly active antiretroviral therapy (HAART) in lowering the plasma viral load in patients with HIV -1 infection and in slowing down the disease progression, many obstacles still remain, including the generation of drug-resistant viral species, the difficulty in eradicating latent reservoirs and the high costs of the therapy. The development of complementary strategies to completely eradicate or to control HIV-1 infection without daily drug intake is therefore a priority. In this context, anti HIV-1 gene therapy based on intracellular immunization of autologous T cells or their progenitors, i.e. the CD34+ hematopoietic stem cells, resistant to infection, appears a particularly promising approach to repopulate the immune system.In order to interfere with different steps of viral replication, we previously developed a series of self-inactivating lentiviral vectors expressing multiple small interfering (si)-RNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1). The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Two effective anti- HIV combinatorial vectors that conferred protection against R5 and X4 tropic virusesin human primary CD4+ T lymphocytes were identified. Further modification of the combinatorial vectors were accomplished by the inclusion of a membrane-anchored peptide (maC46), which has been shown to efficiently block the entry of both CXCR-4 and CCR5-using viruses. The maC46 was inserted under the transcriptional control of the human Elongation Factor 1 promoter. Additionally, an optimized version of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE*) was also inserted in the new generation of lentiviral vectors. The antiviral activity of the optimized vectors was assessed in different cellular models. Overall our results contribute to gain further insights in the design of combinatorial gene therapy approaches against HIV-1 for clinical application.