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Abstract The compelling need to provide adoptive cell therapy (ACT) to an increasing number of oncology patients within a meaningful therapeutic window makes the development of an efficient, fast, versatile, and safe genetic tool for creating recombinant T cells indispensable. In this study, we used nonintegrating minimally sized DNA vectors with an enhanced capability of generating genetically modified cells, and we demonstrate that they can be efficiently used to engineer human T lymphocytes. This vector platform contains no viral components and is capable of replicating extrachromosomally in the nucleus of dividing cells, providing persistent transgene expression in human T cells without affecting their behavior and molecular integrity. We use this technology to provide a manufacturing protocol to quickly generate chimeric antigen receptor (CAR)–T cells at clinical scale in a closed system and demonstrate their enhanced anti-tumor activity in vitro and in vivo in comparison to

Amersham bioscienceD trono addgeneTexmacs miltenyi biotecMiltenyi biotecLumikine xpressJackson immunoresearchMaxcyte gtxPanreac applichemDeutsches krebsforschungszentrumElevator pitch programme of the national centerBd sciencesDrug administrationAgilent technologiesProof of concept programDeutsches krebsforschungszentrum german cancer research centerNature technology corporation