Sustained and specific multiplexed immune checkpoint modulation in CAR T cells induced by targeted epigenome editing.

Engineered T cells equipped with a chimeric antigen receptor (CAR) have shown tremendous clinical success, but tumor-mediated stimulation of T cell inhibitory receptors leads to exhaustion, hampering durable remission in patients. Mitigation of this effect via checkpoint inhibition or genome editing to knockout the genes encoding for these receptors has shown promise. Yet, the side effects of these procedures require better alternatives. Targeted epigenome editing offers a potent strategy to alter gene expression without DNA modifications. Its hit-and-run mechanism enables durable, multiplexed modulation of gene expression with greater safety. Here, we describe multiplexed epigenome editing inactivation of two critical-exhaustion-related genes, and , both in primary human T cells and in prostate-cancer-specific CAR T cells. Epigenetically modified CAR T cells are indistinguishable from parental cells across a range of functional assays. Although the model does not fully mimic T cell exhaustion, limiting functional assessment, gene silencing remains durable across multiple divisions and repeated CAR stimulations. Furthermore, transcriptomic analysis revealed minimal off-target effects not directly attributable to the effectors used. We demonstrate that targeted epigenome editing is effective and safe for multiplexed gene inhibition and holds potential in engineering CAR T cells with enhanced and customizable features.