Alena A. Chekmasova, Marlene S. Strayer, Carmen N. Nichols and David S. Strayer

Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania.

Abstract

In these studies, we tested the effectiveness of combinatorial gene delivery to CCR5-expressing cell lines and primary cells to enhance resistance to HIV-1 infection. The transgenes used were chosen both to decrease membrane CCR5 and to inhibit HIV-1 replication, and were delivered using Tag-deleted SV40-derived vectors. rSV40s are very effective in transducing primary T cells and T cell lines. SV(RNAiR5), SV(RevM10.AU1) and SV(RNAiR5/RevM10.AU1) respectively deliver a small interfering RNA (siRNA) against CCR5, RevM10, to which a C-terminal AU1 epitope was added and which inhibits HIV-1 Rev, or both transgenes together. We used human cell lines (SupT1 and SupT1/CCR5) and primary human monocyte-derived macrophages (MDMs). We assessed RevM10 expression by flow cytometry (FACS). After transduction, we confirmed expression of each transgene by flow cytometry (FACS). Simultaneous delivery of both transgenes in a bifunctional vector protected both SupT1/CCR5 cells and MDMs from R5 tropic HIV-1Ba-L, better than either the monofunctional vectors, SV(RNAiR5) and SV(RevM10.AU1) individually. The bifunctional vector also protected from X4-tropic HIV-1, comparably to SV(RevM10.AU1). SV(RNAiR5) did not protect from X4-tropic HIV-1. Thus, combining these two transgenes in one vector protected from X4-tropic HIV-1 and provided enhanced protection for CCR5-bearing cells from R5-tropic strains of HIV-1. Combinatorial genetic therapy, by targeting of more then one HIV-1 function, may provide effective inhibition of HIV-1 replication.