Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells

Adoptive cellular therapy using genetically engineered immune cells holds tremendous promise for the treatment of advanced cancers. While the number of available receptors targeting tumor specific antigens continues to grow, the current reliance on viral vectors for clinical production of engineered immune cells remains a substantial bottleneck limiting translation of promising new therapies. Here, we describe an optimized methodology for efficient CRISPR-Cas9 based, non-viral engineering of primary human T cells that overcomes key limitations of previous approaches. By synergizing temporal optimization of reagent delivery, reagent composition, and integration mechanism, we achieve targeted integration of large DNA cargo at efficiencies nearing those of viral vector platforms with minimal toxicity. CAR-T cells generated using our approach are highly functional and elicit potent anti-tumor cytotoxicity in vitro and in vivo. Importantly, our method is readily adaptable to current Good Manufacturing Practices (cGMP) and clinical scale-up, offering a near-term alternative to the use of viral vectors for production of genetically engineered T cells for cancer immunotherapy. Overall design: Peripheral Blood T cells from 3 donors were isolated, and cultured overnight. Cells were then stimulated with anti-CD3/anti-CD28 Dynabeads for 24, 36, 48, 72, and 96 hours. A unstimulated control was cultured for 24 hours before harvest. RNA was isolated from samples and used for RNAseq