582. CNS Gene Delivery Targeting CCR3 Protects Brain Microglia from HIV-1

Abstract

Top of pageAbstract Infection by human immunodeficiency virus (HIV-1) requires virus binding to human CD4 plus a coreceptor. CCR5 and CXCR4, are the primary coreceptors used by most HIV-1 isolates. The eotaxin receptor (CCR3) has also been implicated as a coreceptor for HIV infection, particularly in the microglial cells of the brain. This study focuses on the role of CCR3 in CNS HIV-1 infection and therefore as a target for manipulation gene transfer. Two approaches were applied to targeting CCR3. First an anti- CCR3 hybridoma, 5E8-G9-B4, was used to develop a single chain antibody (SFv) that contained the immunoglobulin variable heavy (VH) and variable light (VL) chain. Using overlapping RT-PCR techniques with hybridoma mRNAs, the two V region coding sequences, separated by a flexible hinge region, were cloned into a mammalian expression vector. We also tested a panel of interfering RNA's designed to target a sequence shared by mouse and human CCR3 transcripts. Both approaches were used to test the effectiveness of CCR3 downregulation and consequent protection from M-tropic strains of HIV-1. Cells were treated by adding soluble siRNAs and by transfecting with an expression construct carrying the SFv cDNA. Transfection efficiencies were measured using a marker gene. Of a panel of CCR3 siRNAs, R3-526, and SFv addition to cultures resulted in 60-80% decrease in CCR3 expression when tested in U87.CD4.CCR3 cells, primary human microglia derived from fetal brain and monocyte derived macrophages (MDM). CCR3 downregulation was measured using FACS and immunostaining. Both the SFv and siRNA also significantly decreased (>70%) productive HIV-1 infection, as measured by p24 HIV infection in microglia cells when tested using two different CCR3-tropic HIV-1 isolates: HIV-1ADA and a primary brain isolate. Strains of HIV-1 that utilize only CCR5 as a coreceptor were not inhibited. Because of their effectiveness in transducing microglial cells, rSV40 vectors carrying these moieties are being prepared. Studies involving transduction of these anti-CCR3 moieties, both to assess their effectiveness in downregulating CCR3 and protecting from CCR3- tropic strains of HIV-1, are in progress. Since CCR3 and CCR5 are expressed by microglial cells, therapies targeting both may be needed in treating CNS AIDS. Utilization of CCR3 by these HIV-1 isolates and their susceptibility to inhibition by anti-CCR3 siRNA and SFv in primary brain microglia/macrophages may both help clarify the role of CCR3 as a coreceptor in CNS establishing HIV-1 and provide useful therapeutic insights.