Epigenetic Editing Balances TCR Suppression and Persistence in CAR T cells

Allogeneic chimeric antigen receptor (CAR) T cell therapies offer a scalable, off-the-shelf option for cancer treatment, but their clinical use is limited by the risk of graft-versus-host disease (GvHD), mediated by the endogenous T cell receptor (TCR). Conventional strategies to eliminate TCR expression rely on genome editing tools such as CRISPR/Cas9 or base editing, which introduce permanent DNA changes and pose safety concerns. Here, we present an epigenetic editing approach that enables efficient, specific, and reversible silencing of the CD3e gene, a critical component of the TCR complex, without altering the genome. Through systematic optimization of the epigenetic editor and guide RNA design, we achieved robust TCR silencing in primary T and CAR T cells while preserving CAR expression, activation, and effector function. Transcriptome analysis confirmed minimal off-target effects. In vivo observation suggest the epigenetically silenced T cells to prevent GvHD while persisting longer than TCR-knockout cells, supporting the notion that transient TCR suppression may help balance safety and long-term efficacy. Our findings establish epigenetic editing as a non-genotoxic alternative to genome editing, offering a flexible and safer route to generate next-generation allogeneic CAR T cells. Overall design: RNA-seq profiling of human T cells samples from the same donor which were either untreated, mock electroporated, CRISPR/Cas9 TRAC knocked out or CD3E epigenetically silenced