PROJECT SUMMARY/ABSTRACT Modification of autologous T cells with chimeric antigen receptor (CAR) molecules was first proposed nearly 30 years ago as a therapy for people living with HIV. Since then, CAR-T cells have emerged as a potent and highly successful therapy for liquid tumors, while HIV-specific CAR-T cells have only begun to show efficacy in large animal models and clinical trials. Based on our longstanding interest in CAR-T cell therapies for HIV, we posit three primary barriers that limit the curative potential of this approach. First, low levels of HIV-1 antigen at the cell surface (namely Env protein), especially during antiretroviral therapy (ART), render latently infected cells nearly invisible to CAR-T cells and other virus-specific immune effectors. Second, the wealth of knowledge regarding cellular trafficking of the viral Env protein has yet to be thoroughly applied in the context of Env- dependent HIV cure strategies. Third, CAR-T cell persistence and function wane over time, prior to complete clearance of the latent HIV reservoir. Our groundbreaking preliminary data outlines a path to overcome these limitations. We recently reported findings in four rhesus macaques that were infected with an HIV-like virus, suppressed by ART and then infused with virus-specific CAR-T cells containing the CD4 extracellular domain (CD4CAR). To expand these potent antiviral effectors in vivo, animals were next boosted with an irradiated cell line stably expressing HIV-1 Env. Following ART treatment interruption (ATI), viral control was observed in 2 of 4 animals, consistent with robust and Env-dependent expansion of CD4CAR-T cells. The central goal of this proposal is to increase the potency and feasibility of this approach. In AIM 1, we will transition our Env boosting strategy from an immortalized cell line to an FDA-approved mRNA lipid nanoparticle (mRNA-LNP) platform, analogous to the Moderna and Pfizer/BioNTech vaccines for SARS-CoV-2. Env immunogens will be optimized for CD4CAR T cell interactions and developed as Env mRNA-LNP vaccines. In AIM 2, we will use CRISPR- Cas9 gene editing to extend the durability and function of CD4CAR-T cells. We will compare a series of CAR products that carry inactivated immune checkpoint alleles, which we hypothesize will support more durable function and efficiently clear persistent viral reservoirs. In AIM 3, we will benchmark Env mRNA-LNP and immune checkpoint gene editing strategies in our vetted nonhuman primate (NHP) model of HIV gene therapy. These experiments will feature a powerful competitive repopulation study design, providing critical information on basic CAR biology that cannot be gathered in clinical studies. Together, these aims build on what we believe to be the most promising anti-HIV cell and gene therapy approach reported to date. Our unique and highly informative NHP model of HIV persistence and CAR-T cell therapy will fill in critical gaps in knowledge regarding CAR-T cell safety and fu...