ABSTRACT By harnessing the power of the immune system to attack cancer cells, adoptive T cell therapy (ACT) holds tremendous potential to overcome the limitations of traditional cancer therapies and provide more effective and personalized treatment options for patients. However, the effectiveness of ACT is hindered by T cell exhaustion and the depletion of stem cell memory T cells (TSCM), highlighting the need to improve T cell phenotypes. In several ACT trials, positive responses were closely linked with specific transcriptomic and epigenetic profiles of CD8 T cell subsets in the manufactured T cell product. Epigenetic reprogramming of exhausted cells has been widely postulated as a promising strategy to improve ACT, but we currently lack the knowledge of which genes and epigenetic programs to manipulate. Our long-term goal is to improve cancer treatment outcomes through epigenetic programming of immune cells. To accomplish this goal, we have assembled an experienced and multidisciplinary team with the relevant expertise to address the critical technological challenges and develop next-generation ACT. The overall objective of this project is to translate epigenome editing technologies to enable next-generation ACT through the programming of critical epigenetic nodes that govern complex T cell phenotypes. The central hypothesis is that discovery and targeted perturbation of critical epigenetic nodes can improve T cell function and tumor control. We will accomplish our objective by 1) prioritizing lead targets via high- throughput epigenetic perturbation of primary human T cells, 2) identifying key transcription factors and noncoding regulatory regions driving T cell exhaustion, and 3) investigating the combined effect of master regulatory factors in complex T cell phenotypes. This proposal is innovative because it translates recent developments in epigenome editing technologies and high-throughput targeted epigenetic screening to develop precision interventions that address a fundamental limitation of effective ACT. Collectively, this work will establish a framework for epigenetic engineering in primary human T cells, providing insights into T cell regulation and advancing the development of an enhanced ACT platform.