PROJECT SUMMARY/ABSTRACT Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable clinical responses in patients with B cell malignancies. However, approximately 50% of treated patients do not exhibit durable responses and CAR T cells for solid tumors have been largely ineffective. Poor T cell fitness, whereby T cells lose the ability to expand, persist, and mediate antitumor responses, is a major barrier to progress for the development improved CAR T cell therapies. Emerging research suggests poor fitness is associated with epigenetic changes that lock T cells into a dysfunctional state, and further, that epigenetic reprogramming is mechanistically important for overcoming poor fitness. This presents a unique challenge since existing epigenome engineering approaches 1) are associated with technical hurdles that limit feasibility and translatability, and 2) are either non-specific (e.g., epigenetic drugs that induce global chromatin remodeling) or exceedingly specific (e.g., dCas9-based methods that target 1 or a few genes) and are therefore suboptimal for inducing the extensive but pathway-specific epigenetic changes associated with changes in fitness. The goal of this proposal is to develop and optimize a new class of epigenetic reprogramming factors (ERFs) which can target hundreds, thousands, or tens of thousands of DNA regions in a pathway-specific manner. The ERF platform is modular and fully programmable, thereby enabling fine-tuning of the specificity, strength, and durability of epigenetic marks. We will leverage this technology to target T cell exhaustion and aging-associated dysfunction, which are characterized by stable epigenetic marks that limit T cell responsiveness. ERFs will be designed to prevent or reverse epigenetic signatures that manifest during exhaustion and aging, thereby augmenting CAR T cell fitness and antitumor function. The proposed work will advance the state-of-the-art in T cell reprogramming and provide a powerful approach to enhance CAR T cell potency for liquid and solid tumors. In addition to their translational potential, ERFs can also function as molecular tools to interrogate transcription factor biology, gene expression programs, and epigenetics, and therefore have important implications for basic human T cell biology. Ultimately, we envision an ERF toolkit whereby combinations of ERFs can be tailored to enhance T cell fitness in a patient- or disease-specific manner. Since chromatin remodeling underpins cellular reprogramming in other cell types and disease settings, we predict that ERFs will be broadly applicable to other therapeutic modalities and represent a paradigm shift in the growing field of epigenome engineering.