A versatile one-shot high-multiplex base editing strategy enables the generation of 20-plex knockout CAR T cells with enhanced anti-tumor efficacy

Advances in genomic editing have expanded the therapeutic potential of cell therapies such as chimeric antigen receptor (CAR) T cells, and integrating multifunctional enhancements and allogeneic properties into next-generation therapeutic products requires high-multiplex gene editing strategies that minimize editing-associated toxicity. Here, we pioneered a viral vector-free, one-shot adenine base editing (ABE) approach to enable the simultaneous high-multiplex knockout of at least 20 distinct genes in primary human T cells. The incorporation of this strategy into CAR T cells produced cells with a hyperfunctional phenotype based on a rationally selected 20-plex knockout gene-set. Refinement of this gene-set attenuated initial in vivo toxicity, revealing superior in vivo tumor control of 20-plex edited CAR T cells compared to mock-edited controls in a mouse model of pancreatic ductal adenocarcinoma. We further extended this approach to primary human hematopoietic stem cells (HSCs) and natural killer (NK) cells, demonstrating the method's broad utility for ex vivo gene editing at unprecedented multiplexity levels across diverse cell types of translational relevance. Overall design: RNA-seq profiling of primary human T cells with or without high-multiplex editing of 20 distinct genes (CD3E, B2M, CIITA, PVR, CD52, ICAM1, CD58, PDCD1, CTLA4, CD5, RASA2, MED12, ZC3H12A, RC3H1, TET2, ARID1A, SMARCC1, SMARCA4, SMYD3, ADORA2) 48hrs after electroporationrealized by 2 different gene editing strategies (wildtype Cas9 mRNA or ABE8e mRNA).