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 alternatives. Targeted epigenome editing is a potent strategy to alter gene expression in the absence of DNA modifications. The hit-and-run feature enables durable multiplexed modulation of gene expression with greater safety. Here we describe multiplexed epigenome editing inactivation of two critical exhaustion-related genes, PDCD1 and LAG3, both in primary human T cells and in prostate cancer specific CAR T cells. Epigenetically modified CAR T cells are fully functional and nearly indistinguishable from parental cells in a variety of functional assays. Target gene silencing is durable over multiple cell divisions and repeated CAR-mediated stimulations. Furthermore, transcriptomic analysis revealed minimal off-target effects not directly attributable to the effectors used. We demonstrate that targeted epigenome editing is an effective and safe procedure for multiplexed gene inhibition and that this strategy can be used to engineer CAR T cells with novel desirable features.