Anti-tat Hutat2:Fc Gene Modified Human Monocyte-derived Macrophages Mediated Protection Against Tat-induced Neurotoxicity And HIV-1 Replication: An Novel Protective Approach Against HIV-associated Neurocognitive Disorder

Background: HIV-1 Tat is essential for HIV replication and is also a well-known neurotoxic factor causing HIV-associated neurocognitive disorder(HAND). Currently, combined antiretroviral therapy targeting HIV reverse transcriptase or protease cannot prevent the production of early viral proteins, especially Tat, once HIV infection has been established. HIV-infected macrophages and glial cells in the brain still release Tat into the extracellular space where it can exert direct and indirect neurotoxicity. Therefore, stable production of anti-Tat antibodies in the brain would neutralize HIV-1 Tat and thus provide an effective approach to protect neurons. We constructed a humanized anti-Tat Hutat2:Fc fusion protein with the goal of antagonizing HIV-1 Tat and delivered the gene into human cell lines and primary human monocyte-derived macrophages(hMDM) by an HIV-based lentiviral vector. The function of the anti-Tat Hutat2:Fc fusion protein and the potential side effects of lentiviral vector-mediated gene transfer were evaluated in vitro. Mononuclear phagocytes are currently tested as ideal drug carriers and gene therapy delivery vehicles into the CNS, exploiting their ability to pass freely through the blood brain barrier. Such noninvasive therapeutic approach requires better understanding in the disease homing efficiency of these ex vivo manipulated, adoptively transferred monocytes upon their reintroduction into the blood circulation. This study was set to investigate he homing efficiency of ex vivo manipulated bone marrow-derived monocyte(BMDM) using an acute CNS inflammation mouse model triggered by intracranial injection of E. coli Lipopolysaccharide. Methods: HIV-based defective lentiviral vectors were constructed to express anti-HIV-1 Tat scFv:Fc(Hutat2:Fc fusion protein) and EGFP as an indicator. High titer vectors were generated through transient transfection of 293 T packaging cells, and tested for transduction of human neuronal cell line HTB-11, monocytic cell line U937, as well as primary human peripheral blood monocytes-derived macrophages(hMDM). Expression and secretion of Hutat2:Fc in transduced cells was detected using qRT-PCR, optimized ELISA, Western bloting, and immunofluorescent staining. Dot-immunobinding assay, Western bloting, neuroprotection and HIV challenge assays were performed to assess the biological function of Hutat2:Fc. Potential adverse effects for lentiviral vector-mediated transduction of cells were evaluated by assessing the changes of cell morphology, proliferation, and cellular activation in 15 transcriptional profiling of macrophage-related functional and regulatory genes, and in the releasing of pro-inflammatory cytokines IL1β, IL8, IL10 and TNF-α in transduced hMDM culture mediums. The infiltration of ex vivo SPIO labled mice BMDM or BMDM from GFP transgene mice into the CNS was evaluated using an LPS induced acute CNS inflammation mouse model. Results: Our study demonstrated that HIV-1-based lentiviral vector could transduce cell lines and primary hMDM with high efficiency(99%,95% and 53% for HTB-11, U937 and hMDM, respectively). Gene transduction resulted in a high-level, stable expression of anti-HIV-1 Tat Hutat2:Fc in human neuronal and monocytic cell lines, as well as in primary hMDM. Hutat2:Fc was detectable in both cells and supernatants as a correct format, and continued to accumulate to high levels within supernatants. Hutat2:Fc protected mouse cortical neurons against HIV-1 Tat86 induced neurotoxicity. In addition, both secreted Hutat2:Fc and transduced hMDM led to reducing HIV-1BaL viral replication in human macrophages. Moreover, lentiviral vector-based gene introduction did not result in any significant changes in cytomorphology and cell viability. Although the expression of IL8, STAT1, and IDO1 genes was up-regulated in transduced hMDM, such alternation in gene expression did not affect the neuroprotective effect of Hutat2:Fc. Large amount of adoptive transferred mice BMDM showed up in the inflamed regions within the CNS, peaked at 48 h post injection, and quickly decreased as the inflammation subdued. Conclusions: Our study demonstrated that lentivirus-mediated gene transfer could efficiently deliver the Hutat2:Fc gene into primary h MDM and does not lead to any significant changes in hMDM immune-activation. The neuroprotective and HIV-1 suppressive effects produced by Hutat2:Fc were comparable to that of a full-length anti-Tat antibody. Mice BMDM could infiltrate into the inflammatory hemisphere of the LPS induced acute encephalitis mouse model. This study provides the foundation and insights for future research on the potential use of Hutat2:Fc as a novel gene therapy approach for HAND through utilizing monocytes/macrophages, which naturally cross the blood-brain barrier, for gene delivery.