title: Minicircle-Based Engineering of Chimeric Antigen Receptor (CAR) T Cells.
abstract: Plasmid DNA is being used as a pharmaceutical agent in vaccination, as well as a basic substance and starting material in gene and cell therapy, and viral vector production. Since the uncontrolled expression of backbone sequences present in such plasmids and the dissemination of antibiotic resistance genes may have profound detrimental effects, an important goal in vector development was to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle (MC) DNA. The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform enabling a close-to-random profile of genomic integration. In combination, the MC platform greatly enhances SB transposition and transgene integration resulting in higher numbers of stably modified target cells. We have recently developed a strategy for MC-based SB transposition of chimeric antigen receptor (CAR) transgenes that enable improved transposition rates compared to conventional plasmids and rapid manufacturing of therapeutic CAR T cell doses (Monjezi et al. 2016). This advance enables manufacturing CAR T cells in a virus-free process that relies on SB-mediated transposition from MC DNA to accomplish gene-transfer. Advantages of this approach include a strong safety profile due to the nature of the MC itself and the genomic insertion pattern of MC-derived CAR transposons. In addition, stable transposition and high-level CAR transgene expression, as well as easy and reproducible handling, make MCs a preferred vector source for gene-transfer in advanced cellular and gene therapy. In this chapter, we will review our experience in MC-based CAR T cell engineering and discuss our recent advances in MC manufacturing to accelerate both pre-clinical and clinical implementation.
authors.affiliation: Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hudecek
authors.affiliation: Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany.
authors.forename: Tea
authors.initials: T
authors.lastname: Gogishvili
authors.affiliation: Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany.
authors.forename: Razieh
authors.initials: R
authors.lastname: Monjezi
authors.affiliation: Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Wegner
authors.affiliation: PlasmidFactory, Bielefeld, Germany.
authors.forename: Ram
authors.initials: R
authors.lastname: Shankar
authors.affiliation: PlasmidFactory, Bielefeld, Germany.
authors.forename: Christa
authors.initials: C
authors.lastname: Kruesemann
authors.affiliation: Division of Medical Biotechnology, Paul-Ehrlich-Institute, Langen, Germany.
authors.forename: Csaba
authors.initials: C
authors.lastname: Miskey
authors.affiliation: Division of Medical Biotechnology, Paul-Ehrlich-Institute, Langen, Germany.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Ivics
authors.affiliation: PlasmidFactory, Bielefeld, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Schmeer
authors.affiliation: PlasmidFactory, Bielefeld, Germany. martin.schleef@plasmidfactory.com.
authors.forename: Martin
authors.initials: M
authors.lastname: Schleef
mesh_terms: D000818:Animals; D004251:DNA Transposable Elements; D015972:Gene Expression Regulation, Neoplastic; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D009369:Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000628:therapy*; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism / Q000637:transplantation*; D014162:Transfection; D019076:Transgenes; D019895:Transposases / Q000235:genetics / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D011948:Receptors, Antigen, T-Cell; D019895:Transposases; C494471:sleeping beauty transposase, human
keywords: Chimeric antigen receptor T cell; Immunotherapy; Minicircle; Sleeping Beauty; Transposition