Large scale minicircle-DNA production becomes reality

Background: Plasmid DNA is commonly used in vaccination, gene- or cell therapy but also as a basic substance in viral vector production. The presence of antibiotic (AB) resistance sequences within vectors may result in detectable traces of these e.g. within deriving virus preparations. Especially for safety reasons it is necessary to avoid such sequence elements. Therefore, the European authority for the evaluation of medical products (EMEA) proposes in the guidelines for medical gene transfer products to avoid selection markers like resistances against antibiotics (CPMP/BWP/3088/99). The AB cassette (and other systems to select for the presence of plasmids within the host cell) is used in cultivation to serve as a selection marker. Deleting such structural element results in partial loss of plasmid carrying cells being overgrown by "empty" cells due to their higher specific growth rate. We currently develop a large-scale production process for non-viral DNA vectors without genes for the resistance against antibiotics and other selection markers: Minicircle-DNA. Methods: This vector system is based on a parental plasmid (PP) carrying a selective marker, the origin of replication (ori) for replication within the E. coli host cells, two recombination sites for cis-recombination and the "gene-of-interest" with all necessary elements for regulation in the target cells. The enzyme catalyzed recombination of a PP results in two circular supercoiled molecules: a mini-plasmid (MP) containing the selection marker, the ori and all the unwanted and unessential bacterial sequences and the Minicirde (MC) with nothing but the gene-of-interest and a tiny residual sequence stretch resulting from the recombination. A sequence specific affinity chromatography is used to specifically separate the mini-plasmid from the MC as the basis for a large-scale purification process to manufacture this molecule for pre-clinical and clinical applications. Results: Within this study reporter genes for different types of analyses within various tissues, cells, animals and for testing the mode of administration were used. THe obtained results shown here demonstrate a significant increase of gene expression using equimolar amounts of either plasmid or Minicircle DNA.