Defective lentiviral vector mediated gene transfer of sTNFRS into primary mouse BMDM : a novel therapy for neuroaids

Abstract

Current approaches to the control of human immunodeficiency virus type one (HIV-1) rely largely on the use of highly active antiretroviral therapy (HAART); however, this treatment has limited effect on HIV-1 in the central nervous system (CNS) which lead to HIV-1 associated neurocognitive disorders (HAND) due to the poor migration efficiency of the antiretroviral drugs across the blood brain barrier (BBB). Therefore, new therapeutic approaches for HAND are urgently needed. Since monocyte derived macrophages (MDM) have natural migratory properties that allow them to cross the BBB it has been proposed that the MDM can act as a cellular vehicle for targeted CNS gene delivery. The goal of this research was to establish a method for stable transduction of a soluble tumor necrosis factor-α decoy (sTNFR) into primary mouse bone marrow MDM (BMDM) and so it is hypothesized that constitutive expression of sTNFR following a successful transmigration of these genetically modified cells into the brain would protect neurons from the toxic effects of HIV-1 induced cytokine, tumor necrosis factor-alpha (TNF-α). In order to establish an effective method for highly efficient transduction of primary mouse BMDM, experimental conditions for consistent isolation and cultivation of primary BMDM was optimized in this study. High titer defective lentiviral vector (DLV) stocks expressing sTNFR were produced in HEK 293T and used to transduce primary BMDM. Successful transduction of DLV-sTNFR into primary cultures of BMDM was verified by RT-PCR. The resulting secreted product of the DLV transduced primary BMDM was examined for sTNFR expression by ELISA and showed a stable expression over 43 days. Transduction of BMDM with DLV did not induce TNF-α production as compared with non-transduced BMDM. Gene expression analysis suggested a few up-regulated translation and protein degradation genes due to DLV transduction. Future studies on optimizing DLV mediated gene transfer into primary BMDM to increase sTNFR expression levels for neutralizing TNF-α is needed. Overall, these studies lay the groundwork of this novel therapeutic approach for neuroAIDS in which the natural migratory properties of BMDM can be exploited to deliver neuroprotective factors into the CNS, in a non-invasive manner.