Anti-HIV gene therapy strategies combining multiple siRNA with the entry inhibitor

BACKGROUND: Despite the remarkable success of highly active antiretroviral therapy (HAART) in lowering the viral load in patients with HIV infection, an increasing prevalence of resistant viruses, HAART failure, and a significant incidence of serious side effects have provided the impetus to develop complementary therapies using genetic inhibitors of HIV-1 replication. Anti-HIV gene therapy based on engineering autologous T cells or hematopoietic stem cells resistant to HIV infection appears a particularly promising approach. METHODS: Self - inactivating (SIN) lentiviral vectors expressing different combination of siRNA targeting the cellular gene CCR5 as well as the viral genes vif, tat, and rev were generated. The siRNA were either expressed as single transcriptional unit under the control of different human RNA polymerase III promoters (U6, 7SK, H1), or simultaneously, as an extended shRNA. In addition, the HIV membrane-anchored fusion inhibitor maC46, which derives from the highly conserved amino acid sequence of the C-terminal heptad repeat (HR2) domain of gp41, was also included under the transcriptional control of the human elongation factor 1 alpha (EF1 alpha) promoter. RESULTS: Recombinant particles were produced, titrated and the anti-HIV activity of the combinatorial strategy was tested in cell lines as well as in human primary cells. Two out of the developed vectors showed a potent antiviral effect both on CXCR4- and CCR5-tropic HIV strains in vitro and their efficacy in the murine model is currently underway. The contribution of the maC46 on the antiviral activity is under investigation in challenge experiments carried on both cell lines and primary cells. CONCLUSION: Overall our findings contribute to gain further insights in the design of combinatorial anti-HIV-1 platforms in gene therapy approaches for clinical application.