This thesis project aimed to develop chimeric antigen receptors (CARs) capable of durably suppressing the Human Immunodeficiency Virus Type 1 (HIV) replication, by building upon a previous CD4-based CAR that was employed in several clinical trials. We applied lessons learned from cancer-targeting CARs to optimize the CAR vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains, in an effort to determine which components augmented the ability of CD8 T cells to control HIV replication. CD8 T cells expressing the optimized CARs were at least 50-fold more potent in vitro at controlling HIV replication than the original CD4 CAR or TCR-based approaches and substantially better than broadly neutralizing antibody-based CARs. We then utilized a humanized mouse model of HIV infection to demonstrate superior control over HIV replication, better protection of CD4 T cells, and greater CAR T cell expansion with the optimized vectors compared to the original clinical trial vector. Compared to optimized CD4 CARs containing the CD28 costimulatory domain, CARs containing 4-1BB expanded better in vivo in the absence of antigen and resulted in greater control over HIV replication. We found that the CD4 CAR promoted infection of transduced CD8 T cells and employed CCR5 zinc finger nucleases (ZFNs) or a GP41-based fusion inhibitor to protect the CAR T cells. We employed ZFN-pretreated, CAR-transduced CD8 T cells in our mouse models and saw an enrichment of the disrupted alleles in HIV-infected mice compared to mock controls. In humans, a functional cure will require CAR T cells to prevent the spread of HIV following virus reactivation from the latent reservoir. We modeled this scenario in vitro using ART patient T cells and latency reversing agents (LRAs). Preliminary data suggest that CD4 CAR T cells can respond to low levels of antigen produced by resting ART patient cells in the presence of LRAs. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and provide optimism that CAR T cells could help achieve a functional cure
