Abstract
HIV-1 associated dementia remains a significant public health burden. Clinical and experimental research has shown that reduced levels of brain-derived neurotrophic factor (BDNF) may be a risk factor for neurological complications associated with HIV-1 infection. We are actively testing genetically modified macrophages for their possible use as the cell-based gene delivery vehicle for the central nervous system (CNS). It can be an advantage to use the natural homing/migratory properties of monocyte-derived macrophages to deliver potentially neuroprotective BDNF into the CNS, as a non-invasive manner. Lentiviral-mediated gene transfer of human (h)BDNF plasmid was constructed and characterized. Defective lentiviral stocks were generated by transient transfection of 293T cells with lentiviral transfer plasmid together with packaging and envelope plasmids. High titer lentiviral vector stocks were harvested and used to transduce human neuronal cell lines, primary cultures of human peripheral mononocyte-derived macrophages (hMDM) and murine myeloid monocyte-derived macrophages (mMDM). These transduced cells were tested for hBDNF expression, stability, and neuroprotective activity. The GenomeLab GeXP Genetic Analysis System was used to evaluate transduced cells for any adverse effects by assessing gene profiles of 24 reference genes. High titer vectors were prepared for efficient transduction of neuronal cell lines, hMDM, and mMDM. Stable secretion of high levels of hBDNF was detected in supernatants of transduced cells using western blot and ELISA. The conditioned media containing hBDNF were shown to be protective to neuronal and monocytic cell lines from TNF-α and HIV-1 Tat mediated cytotoxicity. Lentiviral vector-mediated gene transduction of hMDM and mMDM resulted in high-level, stable expression of the neuroprotective factorBDNF in vitro. These findings form the basis for future research on the potential use of BDNF as a novel therapy for neuroAIDS.
Highlights
NeuroAIDS is one of the most devastating consequences of HIV-1 infection which incorporates both infectious and degenerative pathophysiologic pathways
This study demonstrated the feasibility of using lentiviral vectors (LVs) to introduce hBDNF gene into MDMs
Human microglial cell line CHME-5, neuroepithelioma cell line HTB-10 and neuroblastoma cell line HTB-11 were transduced with LVs expressing hBDNF under a CMV promoter, and linked to an enhanced green fluorescent protein via the IRES element
Summary
NeuroAIDS is one of the most devastating consequences of HIV-1 infection which incorporates both infectious and degenerative pathophysiologic pathways. Clinical severity ranges from asymptomatic neurocognitive impairment (ANI) and mild neurocognitive disorder (MND) to its most severe form - HIV-associated dementia (HAD) [2]. Despite the improved life expectancy of HIV-1-infected patients due to the advent of highly active antiretroviral therapy Part of this increase may be attributed to poor penetration of HAART drugs into the CNS to eradicate the virus [8] and increased life expectancy of HIV-1-infected patients. The development of HAD is a significant independent risk factor in AIDSrelated mortality [9]
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