PROJECT SUMMARY While chimeric antigen receptor (CAR) T cell therapy has dramatically altered the cancer therapy landscape, the susceptibility of CAR T cells to succumb to exhaustion in response to chronic stimulation by tumor antigen remains a major challenge. Strategies to overcome CAR T cell exhaustion are critically needed and represent a key research priority. Epigenetic gene activating programs have emerged as important facilitators of CAR T exhaustion, as demonstrated by the observation that CAR T cells lacking the DNA-modifying enzyme TET2 persist in the face of chronic antigen stimulation and the previous knowledge that TET2 reverses the gene silencing program induced by DNA methylation. Taken together, these insights suggest that activation of specific genes may be sufficient and necessary to promote exhaustion. This proposal aims to identify such genes, which represent attractive targets for inhibition to generate more durable CAR T cells. Aim 1 focuses on creating the first split-engineered CRISPR/dCas9-guided epigenome editors. These TET-based technologies (seEE) inducibly activate under the control of a small molecule, enabling controllable activation of target genes in a manner that recapitulates the dynamic, inducible nature of epigenetic reprogramming. Aim 2 seeks to identify TET-regulated genes implicated in CAR T cell exhaustion, first by employing a novel inducible TET overexpression construct (seTET) to decipher the dynamic epigenetic landscape of chronically stimulated CAR T cells, and then by leveraging the unique properties of seEE to screen for genes involved in inducing CAR T cell exhaustion. Candidate genes will be targeted for reactivation and the resulting impact on CAR T cell phenotype will be evaluated, revealing actionable targets for inhibition as a strategy to improve the therapeutic efficacy of CAR T cells. The proposed research will result in the introduction of novel epigenome editing technologies and provide key insights into the epigenetics of CAR T cell exhaustion with high translational potential. Importantly, this research will also train an MD/PhD student to become a well-rounded, independent physician-scientist. In conjunction with clinical immersion activities within the Perelman School of Medicine’s Medical Scientist Training Program, opportunities for collaboration and scientific communication both inside and outside of Penn, and specialty-specific career development activities, the research performed under this funding opportunity will prepare the student for a successful career as a physician-scientist focused on the extension of cell-based immunotherapies and epigenetic prognostication strategies for various diseases of the gastrointestinal tract.