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Immunotherapy with T cell modified with gamma-retroviral or lentiviral (LV) vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in clinical trials. However,

the potential for insertional mutagenesis and genotoxicity of viral vectors is a safety concern, and their cost and regulatory demands a roadblock for rapid and broad clinical translation.

Here, we demonstrate that CAR T cells can be engineered through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles (MCs). We analyzed

genomic distribution of SB and LV integrations and show that a significantly higher proportion of MC-derived CAR transposons compared with LV integrants had occurred outside of highly

expressed and cancer-related genes into genomic safe harbor loci that are not expected to cause mutagenesis or genotoxicity. CD19-CAR T cells engineered with our enhanced SB approach

conferred potent reactivity in vitro and eradicated lymphoma in a xenograft model in vivo. Intriguingly, electroporation of SB MCs is substantially more effective and less toxic compared

with conventional plasmids, and enables cost-effective rapid preparation of therapeutic CAR T-cell doses. This approach sets a new standard in advanced cellular and gene therapy and will

accelerate and increase the availability of CAR T-cell therapy to treat hematologic malignancies.

We thank Silke Frenz and Elke Spirk for expertise in performing the mouse experiments, and Christa Kruesemann for cloning of MCs. We thank Nirav Malani and Frederic Bushman for kindly

providing a raw data set for LV insertions in T cells. Razieh Monjezi was supported by a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of

Würzburg. Michael Hudecek is a member of the Young Scholar Program (Junges Kolleg) of the Bavarian Academy of Sciences. This work was supported by grants from the German Cancer Aid (Deutsche

Krebshilfe e.V., Max Eder Program 110313, M.H.) and the IZKF Würzburg (Interdisziplinäres Zentrum für Klinische Forschung, Projekt D-244, M.H.). Csaba Miskey and Zoltán Ivics were supported

by the Center for Cell and Gene Therapy of the LOEWE (Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz) program in Hessen, Germany.

Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany

Division of Medical Biotechnology, Paul-Ehrlich-Institute, Langen, Germany

MH and ZI are co-inventors on a patent application related to this work that has been filed by the University of Würzburg and the Paul-Ehrlich Institute. M Schleef and M Schmeer are

co-inventors on patent applications related to MC-based transposon- and SB-vectors and are employed at PlasmidFactory GmbH & Co. KG, Bielefeld, Germany.

Supplementary Information accompanies this paper on the Leukemia website

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