title: 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.
authors: Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany.
authors: R
authors: R
authors: Monjezi
authors: Division of Medical Biotechnology, Paul-Ehrlich-Institute, Langen, Germany.
authors: C
authors: C
authors: Miskey
authors: Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany.
authors: T
authors: T
authors: Gogishvili
authors: PlasmidFactory, Bielefeld, Germany.
authors: M
authors: M
authors: Schleef
authors: PlasmidFactory, Bielefeld, Germany.
authors: M
authors: M
authors: Schmeer
authors: Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany.
authors: H
authors: H
authors: Einsele
authors: Division of Medical Biotechnology, Paul-Ehrlich-Institute, Langen, Germany.
authors: Z
authors: Z
authors: Ivics
authors: Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany.
authors: M
authors: M
authors: Hudecek
mesh_terms: D000818:Animals; D018941:Antigens, CD19; D060846:Cell Engineering / Q000379:methods*; D018105:DNA, Kinetoplast / Q000627:therapeutic use; D005822:Genetic Vectors / Q000627:therapeutic use; D019337:Hematologic Neoplasms / Q000628:therapy*; D064593:Heterografts; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D008223:Lymphoma / Q000628:therapy; D051379:Mice; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D018941:Antigens, CD19; D018105:DNA, Kinetoplast; D011948:Receptors, Antigen, T-Cell