Project 3: Summary/Abstract The overall goal of Project 3 is to modulate the levels of anti-HIV-1 chimeric antigen receptor (CAR) and broadly neutralizing antibodies (bNAb) modified immune cells by developing the most effective and safe positive and negative selection strategy to (1) achieve a therapeutic level of repopulation and (2) incorporate a safety “kill-switch” to eliminate the genetically engineered anti-HIV-1 immune effector cells in cases of unexpected adverse effects, such as cytokine storm, autoimmune reaction and malignant transformation. The hematopoietic stem cell-based gene therapy approach has shown great promise to achieve an HIV-1 cure. However, one of the major limitations has been the difficulty of achieving the engraftment levels sufficient to provide therapeutic efficacy, in particular for HIV-1 infected patients where intensive myeloablative conditionings would be an unfavorable risk-benefit. Thus, a safe and titratable positive selection strategy is highly desirable to maximize the level of anti-HIV-1 gene engineered immune cells to treat patients with HIV-1 without dangerous intensive myeloablation. Furthermore, it is important to incorporate a safety “kill-switch” procedure to eliminate the genetically engineered anti-HIV-1 immune effector cells based on lessons learned from severe adverse effects in cancer immunotherapy. Therefore, we will develop a negative selection strategy as a safety “kill-switch” to eliminate genetically engineered immune cells. We will identify the most effective and safe selection strategy from (1) knocking down hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression using RNA interference that enables us to effectively enrich or eliminate anti-HIV-1 gene-modified HSPC using clinically available prodrug 6-thioguanine or methotrexate, (2) co-expressing truncated non-functional human epidermal growth factor receptor (huEGFRt), a cell surface marker for a rapid ex vivo positive selection and in vivo negative selection by an FDA-approved anti-EGFR monoclonal antibody Cetuximab (Erbitux) and (3) the P140K mutant form of human O6-methylguanine-DNA-methyltransferase (MGMTP140K) for a positive selection. We hypothesize that a clinically relevant, safe and effective positive and negative selection strategy can be developed by rigorously evaluating our proposed selection strategies for our anti-HIV-1 CAR and scFv-Fc bNAb combining therapies to achieve a cure of HIV disease.