Project summary: Antiretroviral therapy (ART) has been successful at treating HIV infection and allowing people with HIV (PWH) to lead relatively normal lives. Nevertheless, a cure for HIV has remained elusive, and interruption of ART results in rapid viral rebound. HIV persists during therapy by its ability to enter a state of latent infection in CD4 T cells. A key challenge in the field is defining the molecular mechanisms that regulate HIV expression and contribute to maintaining the latent state. HIV expression in latently infected cells is repressed by the formation of heterochromatin at the integrated provirus that restricts access by key activating transcription factors (TFs) and RNAPol2. Latency reversing agents (LRAs) have been developed that target HIV regulating transcriptional pathways. However, most LRAs reactivate only a fraction of the reservoir, suggesting that latency reversal is inefficient with single agents. We have carried out a set of single cell analyses to characterize the transcriptomic and epigenomic states of latently infected cells, leading to the discovery of a latency associated `signature' in primary CD4 T cells. Since then, we have performed a targeted CRISPR screen of latency-associated genes as well as a chemical screen to identify a set of validated novel host cell factors that individually regulate HIV latency. In this proposal we will capitalize on these findings to identify novel combinations of gene knockouts and small molecule inhibitors that can synergize with our validated HIV regulating factors to achieve broad reactivation of the clinical reservoir. To achieve this, we will take a novel experimental approach involving both combination CRISPR/LRA screening and using cutting edge interpretable machine learning tools to define the key drivers of HIV reactivation. Additionally, we will develop a novel lipid nanoparticle platform for RNA-mediated reprogramming of latently infected cells to promote viral reactivation.