Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies

Joseph G Skeate; Emily J Pomeroy; Nicholas J Slipek; Bryan J Jones; Bryce J Wick; Jae-Woong Chang; Walker S Lahr; Erin M Stelljes; Xiaobai Patrinostro; Blake Barnes; Trevor Zarecki; Joshua B Krueger; Jacob E Bridge; Gabrielle M Robbins; Madeline D McCormick; John R Leerar; Kari T Wenzel; Kathlyn M Hornberger; Kirsti Walker; Dalton Smedley; David A Largaespada; Neil Otto; Beau R Webber; Branden S Moriarity

doi:10.1016/j.ymthe.2024.04.024

Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies

Mol Ther. 2024 Apr 16:S1525-0016(24)00241-7. doi: 10.1016/j.ymthe.2024.04.024. Online ahead of print.

Authors

Joseph G Skeate¹, Emily J Pomeroy¹, Nicholas J Slipek¹, Bryan J Jones², Bryce J Wick¹, Jae-Woong Chang¹, Walker S Lahr¹, Erin M Stelljes¹, Xiaobai Patrinostro², Blake Barnes², Trevor Zarecki², Joshua B Krueger¹, Jacob E Bridge¹, Gabrielle M Robbins¹, Madeline D McCormick¹, John R Leerar², Kari T Wenzel², Kathlyn M Hornberger², Kirsti Walker², Dalton Smedley², David A Largaespada¹, Neil Otto², Beau R Webber³, Branden S Moriarity⁴

Affiliations

¹ Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
² Bio-Techne, Minneapolis, MN 55413, USA.
³ Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address: webb0178@umn.edu.
⁴ Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address: mori0164@umn.edu.

PMID: 38627969
DOI: 10.1016/j.ymthe.2024.04.024

Abstract

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

Keywords: NK cells; T cell; cellular therapy engineering; chimeric antigen receptors; engineering pipeline; high-throughput mutant screening; immunotherapy; transposons.