Minicircle-DNA--a safe and efficient vector system for gene therapy and vaccination

Background: Conventional plasmid-DNA (pDNA) used in gene therapy and vaccination can be subdivided into a bacterial backbone and a transcription unit. Bacterial backbone sequences are needed for pDNA production in bacteria. However, for gene transfer application these sequences are dispensable, reduce the overall efficiency of the DNA agent and, most important, represent a biological safety risk. For instance the dissemination of antibiotic resistance genes, as well as the uncontrolled expression of backbone sequences in patients may have profound detrimental effects and unmethylated CpG motifs have been shown to contribute to silencing of episomal transgene expression. Therefore, an important goal in vector development is to produce supercoiled DNA lacking bacterial backbone sequences. Methods: PlasmidFactory's minicircle production technology is based on two processes: 1) An inducible, sequence specific, close to 100% efficient in vivo recombination process and 2) a novel affinity-based chromatographic purification approach for the isolation of the minicircle-DNA. Results: Quantitative real-time PCR analysis, capillary gel electrophoresis and restriction analysis of the recombination products and the minicircle-DNA revealed a recombination efficiency greater than 99.5% and a purity of the isolated minicircle-DNA of more than 98.5%. Results demonstrate that the described technology facilitates the miligram scale production of highly pure minicircle-DNA for application in gene therapy and vaccination as well as e.g. virus production. The presented process is efficient, stable and suitable for further scale-up in industrial large-scale manufacturing. Within this study, reporter genes for different types of analyses within various tissues, cells, animals and for testing the mode of administration are used. Here, we show results demonstrating a significant increase of gene expression of minicircle DNA in comparison with a standard plasmid.