RT Journal Article
SR Electronic
T1 Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.11.12.468427
DO 10.1101/2021.11.12.468427
A1 Beau R. Webber
A1 Matthew J. Johnson
A1 Nicholas J. Slipek
A1 Walker S. Lahr
A1 Anthony P. DeFeo
A1 Joseph G. Skeate
A1 Xiaohong Qiu
A1 Blaine Rathmann
A1 Miechaleen D. Diers
A1 Bryce Wick
A1 Tom Henley
A1 Modassir Choudhry
A1 R. Scott McIvor
A1 Branden S. Moriarity
YR 2021
UL http://biorxiv.org/content/early/2021/11/13/2021.11.12.468427.abstract
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.