Abstract 3765: Combined silencing of <i>MED12</i> and activation of <i>IL2</i> by epigenetic editing enhances CAR T antitumor potency

Abstract Clearance of solid tumors by Chimeric Antigen Receptor (CAR) T cells demands greater antitumor potency and persistence than liquid tumors. Using CRISPR/Cas9 library screens we have shown that targeted deletion of the Mediator complex subunit 12 (MED12) in human primary CAR T cells boosts their antitumor potency, cytokine secretion and proliferation (Freitas, Science 2022). Translation of CRISPR/Cas9-based enhancements to the clinic requires thorough validation and extensive analysis of specificity and genomic integrity over concerns of genotoxicity and chromosomal rearrangements. Alternatively, CRISPRi/a platforms that modulate gene expression by epigenetic reprogramming (Epi-editors) without irreversibly damaging the genome pose lower risk, in principle. Here we optimize an Epi-editing platform based on mRNA delivery of dCas9-KRAB/DNMT3AL silencers (CRISPRi) and dCas9-2XVP64 activators (CRISPRa), resulting in transient editor expression suitable for modulation of gene expression in primary human CAR T cells. We designed and tested 11 sgRNAs targeting the MED12 promoter and TSS, including a 50x CpG island suitable for hypermethylation. We achieved varying levels of gene silencing up to 6 days post-introduction of CRISPRi. Further optimization of the Epi-edited CAR T manufacturing pipeline yielded efficient and persistent downregulation of MED12 resulting in enhanced antitumor potency of CARs targeting solid tumor antigens (e.g. GD2) in vitro and in vivo, compared to non-edited CAR T cells. RNAseq analysis of Epi-MED12 CAR T cells 10 days post Epi-editing showed that their transcriptional profiles is almost identical to that of MED12 knock-out (KO) CAR T cells, indicating potent and long-lasting gene silencing and cellular reprogramming. We have reported that MED12 KO boosts sensitivity of CAR T cells to IL2 by increased AcH3K27 of STAT5 and AP1 binding sites. We reasoned that activation of the IL2 locus via Epi-editing, would propel a feed-forward loop, further enhancing cytokine secretion and proliferation of Epi-MED12 CAR T cells. First, we designed sgRNAs targeting proximal and distal regulatory regions of IL2 and tested their activator capacity in combination with CRISPRa. Some sgRNAs yielded Epi-IL2 CAR T cells able to produce more IL2 only upon activation, without affecting baseline production, potentially critical for mitigating systemic IL2 toxicities. Finally, we combined Epi-Med12 and Epi-IL2 and performed functional, phenotypic and transcriptomic analysis and observed further enhancement of antitumor potency in vitro and in vivo. Our data show that 1) silencing of MED12 without DNA breaks is possible by Epi-editing, 2) Epi-Med12 CAR T cells display enhanced cytokine secretion, proliferation and antitumor potency in vitro and in vivo, comparable to MED12 KO and 3) simultaneous silencing of MED12 with activation of IL2 further enhances antitumor activity of CAR T cells. Citation Format: Frank Lin, Sean Yamada-Hunter, Alexandra Croft, Nicole DeNamur, Emi Tokuda, Molly Libbrecht, Nathaniel Chartrand, Yiyun Chen, Peng Xu, Akiko Seki, Erica Li, Jason Dean, Katie Freitas, Lucas Thompson, Blythe Sather, Crystal Mackall, Elena Sotillo. Combined silencing of MED12 and activation of IL2 by epigenetic editing enhances CAR T antitumor potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3765.