Epigenetic Editing Balances TCR Suppression and Persistence in CAR T cells
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ABSTRACT:
PROVIDER: PRJNA1276116 | ENA |
REPOSITORIES: ENA
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Project description: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 CD3ε 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.
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Project description:Insufficient functional T cell persistence impedes therapeutic success of chimeric antigen receptor (“CAR”) therapies. Here, we performed a CAR-adapted base editing screen of PIK3CD, a key regulator of T cell function, metabolism, and fate. We identified point mutations that beneficially modulate CAR T cell profiles in 4-1BBz and 28z CAR T cells, respectively. Remarkably, point mutations with differing effects on PI3Kδ signaling activity were advantageous in distinct CAR contexts: The PI3Kδ-activating mutation E81K enhanced proliferation, metabolic fitness and effector function in 4-1BBz CARs, promoting long-term functional persistence and enhanced therapeutic efficacy in vivo. Conversely, the PI3Kδ-attenuating mutation L32P improved T cell memory formation and functionality in 28z CAR T cells. Together, our approach of Rational Optimization of Activation-dependent Signaling via Targeted Allelic Reprogramming (ROADSTAR) illustrates the importance of CAR design-specific fine-tuning of tailoring intrinsic T cell signaling and demonstrates the potential of base editing for next-generation cellular therapies. Raw data files not provided due to data sensitivity and privacy concerns.
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