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Minicircle‐DNA production by site specific recombination and protein–DNA interaction chromatography
Author(s) -
Mayrhofer Peter,
Blaesen Markus,
Schleef Martin,
Jechlinger Wolfgang
Publication year - 2008
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1243
Subject(s) - minicircle , biology , dna , plasmid , dna supercoil , site specific recombination , gene , microbiology and biotechnology , recombination , genetics , dna replication , recombinase
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 importantly, represent a biological safety risk. For example, the dissemination of antibiotic resistance genes, as well as the uncontrolled expression of backbone sequences, 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 nonviral vector development is to produce supercoiled pDNA lacking bacterial backbone sequences. Methods A method is described to provide circular, supercoiled minimal expression cassettes (minicircle‐DNA) based on two processes: (i) an inducible, sequence specific, in vivo recombination process that is almost 100% efficient and (2) a novel affinity‐based chromatographic purification approach for the isolation of the minicircle‐DNA. Results Quantitative real‐time polymerase chain reaction 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%. Conclusions The results obtained in the present study demonstrate that the described technology facilitates the production of highly pure minicircle‐DNA for direct application in gene therapy and vaccination. The process described is efficient, stable and suitable for further scale‐up in industrial large‐scale manufacturing. Copyright © 2008 John Wiley & Sons, Ltd.