Enhanced car t-cell engineering using non-viral sleeping beauty transposition from minicircle vectors

ABSTRACT 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. Access through your institution Buy or subscribe This is a preview of

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CLINICAL TRIAL WITH SLAMF7 CAR-T CELLS PREPARED BY VIRUS-FREE _SLEEPING BEAUTY_ GENE TRANSFER TO TREAT MULTIPLE MYELOMA Article Open access 13 April 2021 CAS9-INDUCED TARGETED INTEGRATION OF

LARGE DNA PAYLOADS IN PRIMARY HUMAN T CELLS VIA HOMOLOGY-MEDIATED END-JOINING DNA REPAIR Article 13 December 2023 INSERTING EF1Α-DRIVEN CD7-SPECIFIC CAR AT _CD7_ LOCUS REDUCES FRATRICIDE

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  Google Scholar  Download references ACKNOWLEDGEMENTS 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. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany R Monjezi, T Gogishvili, H

Einsele & M Hudecek * Division of Medical Biotechnology, Paul-Ehrlich-Institute, Langen, Germany C Miskey & Z Ivics * PlasmidFactory, Bielefeld, Germany M Schleef & M Schmeer

Authors * R Monjezi View author publications You can also search for this author inPubMed Google Scholar * C Miskey View author publications You can also search for this author inPubMed 

Google Scholar * T Gogishvili View author publications You can also search for this author inPubMed Google Scholar * M Schleef View author publications You can also search for this author

inPubMed Google Scholar * M Schmeer View author publications You can also search for this author inPubMed Google Scholar * H Einsele View author publications You can also search for this

author inPubMed Google Scholar * Z Ivics View author publications You can also search for this author inPubMed Google Scholar * M Hudecek View author publications You can also search for

this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to M Hudecek. ETHICS DECLARATIONS COMPETING INTERESTS 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. ADDITIONAL INFORMATION Supplementary Information accompanies this paper on the Leukemia website

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KB) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Monjezi, R., Miskey, C., Gogishvili, T. _et al._ Enhanced CAR T-cell engineering using non-viral

_Sleeping Beauty_ transposition from minicircle vectors. _Leukemia_ 31, 186–194 (2017). https://doi.org/10.1038/leu.2016.180 Download citation * Received: 08 February 2016 * Revised: 08 June

2016 * Accepted: 10 June 2016 * Published: 24 June 2016 * Issue Date: January 2017 * DOI: https://doi.org/10.1038/leu.2016.180 SHARE THIS ARTICLE Anyone you share the following link with

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