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CAR T cell generation by piggyBac transposition from linear doggybone™ DNA vectors requires transposon DNA flanking regions.
Molecular Therapy - Methods & Clinical Development ( IF 4.875 ) Pub Date : 2020-01-16 , DOI: 10.1016/j.omtm.2019.12.020
David C. Bishop; Lisa Caproni; Kavitha Gowrishankar; Michal Legiewicz; Kinga Karbowniczek; John Tite; David J. Gottlieb; Kenneth P. Micklethwaite

CD19-specific chimeric antigen receptor (CAR19) T cells generated using viral vectors are an efficacious but costly treatment for B cell malignancies. The non-viral 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 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.
更新日期:2020-01-21

 

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