Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells
Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells
Abstract 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.
Slipek Nicholas J.、DeFeo Anthony P.、Diers Miechaleen D.、Moriarity Branden S.、Rathmann Blaine、Wick Bryce、Skeate Joseph G.、Webber Beau R.、Johnson Matthew J.、Henley Tom、Choudhry Modassir、Scott McIvor R.、Qiu Xiaohong、Lahr Walker S.
Department of Pediatrics, University of Minnesota||Masonic Cancer Center, University of Minnesota||Center for Genome Engineering, University of MinnesotaDepartment of Pediatrics, University of Minnesota||Masonic Cancer Center, University of Minnesota||Center for Genome Engineering, University of MinnesotaDepartment of Pediatrics, University of Minnesota||Masonic Cancer Center, University of Minnesota||Center for Genome Engineering, University of MinnesotaDepartment of Pediatrics, University of Minnesota||Masonic Cancer Center, University of Minnesota||Center for Genome Engineering, University of MinnesotaDepartment of Pediatrics, University of Minnesota||Masonic Cancer Center, University of Minnesota||Center for Genome Engineering, University of MinnesotaDepartment of Pediatrics, University of Minnesota||Masonic Cancer Center, University of Minnesota||Center for Genome Engineering, University of MinnesotaDepartment of Pediatrics, University of Minnesota||Masonic Cancer Center, University of Minnesota||Center for Genome Engineering, University of MinnesotaDepartment of Pediatrics, University of Minnesota||Masonic Cancer Center, University of Minnesota||Center for Genome Engineering, University of MinnesotaDepartment of Pediatrics, University of Minnesota||Masonic Cancer Center, University of Minnesota||Center for Genome Engineering, University of MinnesotaIntima BioscienceIntima BioscienceCenter for Genome Engineering, University of Minnesota||Department of Genetics, Cell Biology and Development, University of MinnesotaDepartment of Pediatrics, University of Minnesota||Masonic Cancer Center, University of Minnesota||Center for Genome Engineering, University of MinnesotaDepartment of Pediatrics, University of Minnesota||Masonic Cancer Center, University of Minnesota||Center for Genome Engineering, University of Minnesota
医学研究方法肿瘤学生物科学研究方法、生物科学研究技术
Slipek Nicholas J.,DeFeo Anthony P.,Diers Miechaleen D.,Moriarity Branden S.,Rathmann Blaine,Wick Bryce,Skeate Joseph G.,Webber Beau R.,Johnson Matthew J.,Henley Tom,Choudhry Modassir,Scott McIvor R.,Qiu Xiaohong,Lahr Walker S..Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells[EB/OL].(2025-03-28)[2025-05-13].https://www.biorxiv.org/content/10.1101/2021.11.12.468427.点此复制
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