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Engineered Zinc Finger Protein Targeting 2LTR Inhibits HIV Integration in Hematopoietic Stem and Progenitor Cell-Derived Macrophages: In Vitro Study.

Siriraj Center for Regenerative Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. Center of Biomolecular Therapy and Diagnostic, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand. Faculty of Medicine, King Mongkut's Institute of Technology Ladkrabang, Bangkok 10520, Thailand. Research Institute for Health Sciences, Chiang Mai University, Chiang Mai 50200, Thailand. Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA. Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Excellent Center for Drug Discovery, Mahidol University, Bangkok 10400, Thailand. Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand. Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand. Center of Innovative Immunodiagnostic Development, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.

International journal of molecular sciences. 2022;(4)
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Abstract

Human hematopoietic stem/progenitor cell (HSPC)-based gene therapy is a promising direction for curing HIV-1-infected individuals. The zinc finger protein (2LTRZFP) designed to target the 2-LTR-circle junction of HIV-1 cDNA was previously reported as an intracellular antiviral molecular scaffold that prevents HIV integration. Here, we elucidate the efficacy and safety of using 2LTRZFP in human CD34+ HSPCs. We transduced 2LTRZFP which has the mCherry tag (2LTRZFPmCherry) into human CD34+ HSPCs using a lentiviral vector. The 2LTRZFPmCherry-transduced HSPCs were subsequently differentiated into macrophages. The expression levels of pro-apoptotic proteins of the 2LTRZFPmCherry-transduced HSPCs showed no significant difference from those of the non-transduced control. Furthermore, the 2LTRZFPmCherry-transduced HSPCs were successfully differentiated into mature macrophages, which had normal phagocytic function. The cytokine secretion assay demonstrated that 2LTRZFPmCherry-transduced CD34+ derived macrophages promoted the polarization towards classically activated (M1) subtypes. More importantly, the 2LTRZFPmCherry transduced cells significantly exhibited resistance to HIV-1 integration in vitro. Our findings demonstrate that the 2LTRZFPmCherry-transduced macrophages were found to be functionally and phenotypically normal, with no adverse effects of the anti-HIV-1 scaffold. Our data suggest that the anti-HIV-1 integrase scaffold is a promising antiviral molecule that could be applied to human CD34+ HSPC-based gene therapy for AIDS patients.