Characterizing piggyBat-a transposase for genetic modification of T cells

Gaurav Sutrave; Ning Xu; Tiffany C Y Tang; Alla Dolnikov; Brian Gloss; David J Gottlieb; Kenneth P Micklethwaite; Kavitha Gowrishankar

doi:10.1016/j.omtm.2022.03.012

Characterizing piggyBat-a transposase for genetic modification of T cells

Mol Ther Methods Clin Dev. 2022 Mar 22:25:250-263. doi: 10.1016/j.omtm.2022.03.012. eCollection 2022 Jun 9.

Authors

Gaurav Sutrave^{1

2}, Ning Xu^{3

4}, Tiffany C Y Tang^{3

4}, Alla Dolnikov^{3

4}, Brian Gloss¹, David J Gottlieb^{1

2

5}, Kenneth P Micklethwaite^{1

2

5

6}, Kavitha Gowrishankar^{1

2}

Affiliations

¹ Westmead Institute for Medical Research, Sydney, NSW, Australia.
² Sydney Medical School, The University of Sydney, Sydney, NSW, Australia.
³ Children's Cancer Institute, University of New South Wales, Sydney, NSW, Australia.
⁴ School of Women's and Children's Health, UNSW Sydney, Sydney, NSW, Australia.
⁵ Blood Transplant and Cell Therapies Program, Department of Haematology, Westmead Hospital, Hawkesbury Road, Westmead, NSW 2145, Australia.
⁶ Blood Transplant and Cell Therapies Laboratory, NSW Health Pathology - ICPMR Westmead, Sydney, NSW, Australia.

Abstract

Chimeric antigen receptor (CAR) T cells targeting CD19 have demonstrated remarkable efficacy in the treatment of B cell malignancies. Current CAR T cell manufacturing protocols are complex and costly due to their reliance on viral vectors. Non-viral systems of genetic modification, such as with transposase and transposon systems, offer a potential streamlined alternative for CAR T cell manufacture and are currently being evaluated in clinical trials. In this study, we utilized the previously described transposase from the little brown bat, designated piggyBat, for production of CD19-specific CAR T cells. PiggyBat demonstrates efficient CAR transgene delivery, with a relatively low variability in integration copy number across a range of manufacturing conditions as well as a similar integration site profile to super-piggyBac transposon and viral vectors. PiggyBat-generated CAR T cells demonstrate CD19-specific cytotoxic efficacy in vitro and in vivo. These data demonstrate that alternative, naturally occurring DNA transposons can be efficiently re-tooled to be exploited in real-world applications.

Keywords: CAR; CD19; T cell immunotherapy; chimeric antigen receptor; non-viral vector; piggybat; transposons.