Plasmid DNA is commonly used in vaccination, gene- or cell therapy but also as a basic substance in viral vector and RNA production. Backbone sequences in a plasmid are only needed for amplification in bacterial cultures. Since the uncontrolled expression of these sequences may have profound detrimental effects, e.g. the dissemination of antibiotic resistance genes, an important goal in vector development was to produce supercoiled DNA lacking such bacterial backbone sequences. Circular, supercoiled DNA that is free of any selection markers, CpG containing backbone structures and even the origin of replication is now available: Minicircle DNA (MC), consisting almost only of (therapeutically) active gene cassette.

MC have proven to be a reliable tool for efficient transgene expression in eukaryotic cells in vitro and in vivo but also for ex vivo modification for cell therapy. MC show a superior performance in CAR-T cell therapy research if carrying a Sleeping Beauty transposon element, 4.4-fold increasing the transposition rate when compared to plasmid vectors due to the reduced size of the backbone (“SCAR”).

Furthermore, the use of MC in AAV manufacturing could demonstrate a significantly improved quality of the resulting AAV and significantly reducing contaminations of AAV capsids carrying undesired plasmid backbone sequences from about 26.1% to below background levels.

Recent trends and progress in pre-clinical studies suggest that the time has come for preparation of such minimalistic vectors in a “High Quality Grade” to enable for example the production of viral vectors for gene therapy.

PlasmidFactory, Bielefeld, Germany (www.plasmidfactory.com) successfully developed a production process, suitable to achieve the upcoming needs in minicircle DNA manufacturing.