HIV associated neurocognitive disorders and lentiviral vector-mediated stable expression of anti-HIV-1 Tat intrabodies in human macrophage, neuronal, and primary peripheral blood mononuclear cells as a potential therapy for neuroAIDS
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2012-05
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University of Hawaii at Manoa
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HIV-1 Tat is required for HIV replication and is also a known potent neurotoxin causing HIV-Associated Dementia. To test whether stable production of secreted Tat antibody in the brain could be an effective approach to inactivate Tat and thus provide protection from neuroAIDS, our research integrates HIV-1 Tat single chain variable fragment (scFv) intrabodies with a novel gene therapy approach utilizing monocytes, which naturally cross the blood-brain barrier, for gene delivery. HIV-based defective lentiviral vectors were constructed to express one of two different HIV-Tat scFv antibodies or control scFvs with a CMV promoter and Fc-fusion protein and GFP as indicator genes. High titer vectors (2x107) were generated through calcium phosphate precipitation mediated transfection of human embryonic kidney 293T packaging cells, and tested for transduction of established human neuroblastoma (HTB-11) and microglial (CHME-5) cells, as well as primary peripheral blood mononuclear cells (PBMC). Expression of anti-HIV-Tat scFv in transduced cell lines was detected using optimized ELISA, Western Blot, and Immunofluorescent staining. Immunoblot and Neuroprotection assays were performed to assess anti-HIV-1 Tat scFv function and multiplex genetic expression analysis of 24 common reference genes was utilized to determine any cellular gene expression changes. Efficient transduction ranging from 80% to 100% in HTB-11 and CHME-5 cell lines, determined by GFP quantification, was achieved at a multiplicity of infection of 10 and confirmed by PCR. Long-term observation of transduced cells revealed no apparent change as compared to normal cells in terms of cell growth and morphology. Multiplex genetic expression analysis revealed similar gene expression levels in non-transduced and transduced cells. The expression of transgenes (GFP and anti-HIV-1 Tat scFv) in transduced cells was stable long term (>20 cell passages) and intracellular production of these genes was confirmed through Immunofluorescent staining. Western Blot assays confirmed anti-HIV-1 Tat scFv expression and ELISA quantitatively assessed secreted anti-HIV-1 Tat scFv concentrations to range from 350ng/mL in transduced CHME-5 to 700ng/mL in transduced HTB-11 cell lines. In addition, Immunoblot assays demonstrated the accurate biological function of secreted anti-HIV-1 Tat scFv by its specific binding to HIV-1 Tat protein in vitro and Neuroprotection assays against HIV-1 Tat and gp-120 demonstrated that anti-HIV-1 Tat scFv, both in transduced cells and conditioned media provided significant protection from both neurotoxins (p<0.01). Primary PBMCs were isolated and transduced at a multiplicity of infection of 10, achieving 10% transduction efficiency as determined by GFP quantification and confirmed by PCR. ELISA detected secreted anti-HIV-1 Tat scFv, which provided significant protection from HIV-1 Tat and Gp120 neurotoxins (p<0.01). Multiplex genetic expression analysis revealed similar gene expression levels in both transduced and non-transduced PBMC cultures. Findings from this study support the in-depth study of anti-HIV-1 Tat scFv, which will facilitate the development and potential use of the constructed lentiviral vectors to deliver anti-HIV-1 Tat scFv into the brain for neuroprotective intervention using genetically modified macrophage cells as a vehicle.
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HIV, neuroAIDS
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Theses for the degree of Master of Science (University of Hawaii at Manoa). Public Health.
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