Background: CCR5 chemokine receptor is important in HIV entry into susceptible cells. CCR5 deletion protects from HIV infection without negative consequences. Thus, CCR5 is an excellent target for elimination by gene therapy. Recombinant SV40 vectors (rSV40) are effective in delivering combination genetic therapies and so were tested for their ability to deliver these anti-CCR5 transgenes: a single-chain antibody, a small interfering RNA (RNAi), and a hammerhead ribozyme against CCR5 to cell lines, to inhibit replication of R5-tropic HIV.

Methods: rSV40 vectors used in these studies carried a CCR5-specific, single-chain antibody driven by CMV immediate early promoter (SV[2C7]), a hammerhead ribozyme and a RNAi, both targeting the 5’ end of human CCR5 mRNA and both being driven by an adenovirus pol III promoter ([SV{VCKA1} and SV[CCR5i], respectively). SupT1 cells stably transduced with human CCR5 DNA (SupT1/R5) were challenged with HIV BaL. HIV replication was measured by ELISA for p24.

Results: SV(2C7), SV(CCR5i), and SV(VCKA1) transduced HIV-1 susceptible T-cell lines without detectable toxicity. By immuno-staining and FACS analysis, > 90% of unselected cells were transduced by SV(2C7). In situ RT-PCR revealed transduction of > 90% unselected cells by SV(CCR5i) and SV(VCKA1). Both ribozyme and RNAi against CCR5 efficiently reduced cell CCR5 mRNA. All transgenes blocked CCR5 expression, as analyzed by immunostaining or FACS. SupT1/R5 cells treated with SV(2C7), SV(CCR5i) and SV(VCKA1) strongly resisted challenge with 0.1 ng HIV BaL. Protection by these transgenes individually was overcome by 1 and 2 ng of HIV BaL, but inhibition of HIV was restored by combined transduction with SV(2C7), SV(CCR5i) and SV(VCKA1).

Conclusions: Reducing the concentration of CCR5 receptor at the cell membrane using SV(2C7), SV(CCR5i) and SV(VCKA1) produced human T-cell lines resistant to HIV R5-tropic infection. These findings suggest combination gene delivery, including RNAi targeting CCR5 can be effectively combined with rSV40 vectors to provide augmented protection from R5-tropic strains of HIV. Combined transduction improved protection: very high challenge doses of HIV BaL overwhelmed protection by single transgenes, but not that conferred by co-transduction with 2 or 3 transgenes. Use of rSV40 vectors readily lends itself to multiple therapeutic combinations or multiple administrations to provide optimum protection where and when very high HIV concentrations may be attained.