Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair

Beau R Webber; Matthew J Johnson; Joseph G Skeate; Nicholas J Slipek; Walker S Lahr; Anthony P DeFeo; Lauren J Mills; Xiaohong Qiu; Blaine Rathmann; Miechaleen D Diers; Bryce Wick; Tom Henley; Modassir Choudhry; Timothy K Starr; R Scott McIvor; Branden S Moriarity

doi:10.1038/s41551-023-01157-4

Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair

Nat Biomed Eng. 2023 Dec 13. doi: 10.1038/s41551-023-01157-4. Online ahead of print.

Authors

Beau R Webber^#^{1

2

3}, Matthew J Johnson^#^{4

5

6}, Joseph G Skeate^{4

5

6}, Nicholas J Slipek^{4

5

6}, Walker S Lahr^{4

5

6}, Anthony P DeFeo^{4

5

6}, Lauren J Mills^{4

5}, Xiaohong Qiu^{4

5

6}, Blaine Rathmann^{4

5

6}, Miechaleen D Diers^{4

5

6}, Bryce Wick^{4

5

6}, Tom Henley⁷, Modassir Choudhry⁷, Timothy K Starr^{5

8

9}, R Scott McIvor^{6

8}, Branden S Moriarity^{10

11

12}

Affiliations

¹ Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. webb0178@umn.edu.
² Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. webb0178@umn.edu.
³ Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. webb0178@umn.edu.
⁴ Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.
⁵ Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
⁶ Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.
⁷ Intima Bioscience, New York, NY, USA.
⁸ Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA.
⁹ Department of Ob-Gyn and Women's Health, University of Minnesota, Minneapolis, MN, USA.
¹⁰ Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. mori0164@umn.edu.
¹¹ Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. mori0164@umn.edu.
¹² Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. mori0164@umn.edu.

^# Contributed equally.

PMID: 38092857
DOI: 10.1038/s41551-023-01157-4

Abstract

The reliance on viral vectors for the production of genetically engineered immune cells for adoptive cellular therapies remains a translational bottleneck. Here we report a method leveraging the DNA repair pathway homology-mediated end joining, as well as optimized reagent composition and delivery, for the Cas9-induced targeted integration of large DNA payloads into primary human T cells with low toxicity and at efficiencies nearing those of viral vectors (targeted knock-in of 1-6.7 kb payloads at rates of up to 70% at multiple targeted genomic loci and with cell viabilities of over 80%). We used the method to produce T cells with an engineered T-cell receptor or a chimaeric antigen receptor and show that the cells maintained low levels of exhaustion markers and excellent capacities for proliferation and cytokine production and that they elicited potent antitumour cytotoxicity in vitro and in mice. The method is readily adaptable to current good manufacturing practices and scale-up processes, and hence may be used as an alternative to viral vectors for the production of genetically engineered T cells for cancer immunotherapies.

Abstract

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