CAR T Cell Generation by piggyBac Transposition from Linear Doggybone DNA Vectors Requires Transposon DNA-Flanking Regions

David C Bishop; Lisa Caproni; Kavitha Gowrishankar; Michal Legiewicz; Kinga Karbowniczek; John Tite; David J Gottlieb; Kenneth P Micklethwaite

doi:10.1016/j.omtm.2019.12.020

CAR T Cell Generation by piggyBac Transposition from Linear Doggybone DNA Vectors Requires Transposon DNA-Flanking Regions

Mol Ther Methods Clin Dev. 2020 Jan 16:17:359-368. doi: 10.1016/j.omtm.2019.12.020. eCollection 2020 Jun 12.

Authors

David C Bishop^{1

2

3

4}, Lisa Caproni⁵, Kavitha Gowrishankar^{1

4}, Michal Legiewicz⁵, Kinga Karbowniczek⁵, John Tite⁵, David J Gottlieb^{1

2

3

4

6

7}, Kenneth P Micklethwaite^{1

2

3

4

6}

Affiliations

¹ Westmead Institute for Medical Research, Sydney, NSW, Australia.
² Department of Haematology, Westmead Hospital, Sydney, NSW 2145, Australia.
³ Blood and Bone Marrow Transplant Unit, Westmead Hospital, Sydney, NSW, Australia.
⁴ Sydney Medical School, The University of Sydney, Sydney, NSW, Australia.
⁵ Touchlight Genetics, Hampton, United Kingdom.
⁶ Sydney Cellular Therapies Laboratory, Westmead Hospital, Sydney, NSW, Australia.
⁷ Department of Medicine, Westmead Hospital, Sydney, NSW, Australia.

Abstract

CD19-specific chimeric antigen receptor (CAR19) T cells, generated using viral vectors, are an efficacious but costly treatment for B cell malignancies. The nonviral piggyBac transposon system provides a simple and inexpensive alternative for CAR19 T cell production. Until now, piggyBac has been plasmid based, facilitating economical vector amplification in bacteria. However, amplified plasmids have several undesirable qualities for clinical translation, including bacterial genetic elements, antibiotic-resistance genes, and the requirement for purification to remove endotoxin. Doggybones (dbDNA) are linear, covalently closed, minimal DNA vectors that can be inexpensively produced enzymatically in vitro at large scale. Importantly, they lack the undesirable features of plasmids. We used dbDNA incorporating piggyBac to generate CAR19 T cells. Initially, expression of functional transposase was evident, but stable CAR expression did not occur. After excluding other causes, additional random DNA flanking the transposon within the dbDNA was introduced, promoting stable CAR expression comparable to that of using plasmid components. Our findings demonstrate that dbDNA incorporating piggyBac can be used to generate CAR T cells and indicate that there is a requirement for DNA flanking the piggyBac transposon to enable effective transposition. dbDNA may further reduce the cost and improve the safety of CAR T cell production with transposon systems.

Keywords: B cell malignancy; CAR; CD19; Chimeric antigen receptor; T cell immunotherapy; doggybone; linear DNA vector; minimal DNA vector; non-viral vector; piggyBac transposon.