PT  - JOURNAL ARTICLE
AU  - Beau R. Webber
AU  - Matthew J. Johnson
AU  - Nicholas J. Slipek
AU  - Walker S. Lahr
AU  - Anthony P. DeFeo
AU  - Joseph G. Skeate
AU  - Xiaohong Qiu
AU  - Blaine Rathmann
AU  - Miechaleen D. Diers
AU  - Bryce Wick
AU  - Tom Henley
AU  - Modassir Choudhry
AU  - R. Scott McIvor
AU  - Branden S. Moriarity
TI  - Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells
AID  - 10.1101/2021.11.12.468427
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.11.12.468427
4099  - http://biorxiv.org/content/early/2021/11/13/2021.11.12.468427.short
4100  - http://biorxiv.org/content/early/2021/11/13/2021.11.12.468427.full
AB  - 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 significant 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 cGMP compliant manufacturing 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.Competing Interest StatementB.R.W., R.S.M, and B.S.M. are principal investigators of Sponsored Research Agreements funded by Intima Biosciences to support the work in this manuscript. Patents have been filed covering the methods and approaches outlined in this manuscript.