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

Beau R. Webber, Matthew J. Johnson, Nicholas J. Slipek, Walker S. Lahr, Anthony P. DeFeo, Joseph G. Skeate, Xiaohong Qiu, Blaine Rathmann, Miechaleen D. Diers, Bryce Wick, Tom Henley, Modassir Choudhry, R. Scott McIvor, Branden S. Moriarity
doi: https://doi.org/10.1101/2021.11.12.468427
Beau R. Webber
1Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
2Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
3Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
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Matthew J. Johnson
1Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
2Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
3Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
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Nicholas J. Slipek
1Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
2Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
3Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
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Walker S. Lahr
1Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
2Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
3Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
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Anthony P. DeFeo
1Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
2Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
3Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
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Joseph G. Skeate
1Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
2Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
3Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
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Xiaohong Qiu
1Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
2Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
3Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
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Blaine Rathmann
1Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
2Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
3Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
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Miechaleen D. Diers
1Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
2Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
3Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
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Bryce Wick
1Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
2Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
3Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
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Tom Henley
4Intima Bioscience, New York, USA
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Modassir Choudhry
4Intima Bioscience, New York, USA
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R. Scott McIvor
3Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
5Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA
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Branden S. Moriarity
1Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
2Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA
3Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA
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  • For correspondence: mori0164@umn.edu
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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.

Competing Interest Statement

B.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.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted November 13, 2021.
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Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells
Beau R. Webber, Matthew J. Johnson, Nicholas J. Slipek, Walker S. Lahr, Anthony P. DeFeo, Joseph G. Skeate, Xiaohong Qiu, Blaine Rathmann, Miechaleen D. Diers, Bryce Wick, Tom Henley, Modassir Choudhry, R. Scott McIvor, Branden S. Moriarity
bioRxiv 2021.11.12.468427; doi: https://doi.org/10.1101/2021.11.12.468427
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Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells
Beau R. Webber, Matthew J. Johnson, Nicholas J. Slipek, Walker S. Lahr, Anthony P. DeFeo, Joseph G. Skeate, Xiaohong Qiu, Blaine Rathmann, Miechaleen D. Diers, Bryce Wick, Tom Henley, Modassir Choudhry, R. Scott McIvor, Branden S. Moriarity
bioRxiv 2021.11.12.468427; doi: https://doi.org/10.1101/2021.11.12.468427

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