Minicircle-DNA production by site specific recombination and protein-DNA interaction chromatography. Export

@article{citeulike:3958578, abstract = {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.}, author = {Mayrhofer, P. and Blaesen, M. and Schleef, M. and Jechlinger, W.}, citeulike-article-id = {3958578}, citeulike-linkout-0 = {http://dx.doi.org/10.1002/jgm.1243}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/18767031}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=18767031}, doi = {10.1002/jgm.1243}, issn = {1521-2254}, journal = {The journal of gene medicine}, keywords = {gene-therapy, plasmid}, month = {November}, number = {11}, pages = {1253--1269}, posted-at = {2009-01-27 12:26:36}, priority = {2}, title = {Minicircle-DNA production by site specific recombination and protein-DNA interaction chromatography.}, url = {http://dx.doi.org/10.1002/jgm.1243}, volume = {10}, year = {2008} }

TY - JOUR ID - citeulike:3958578 L3 - citeulike-article-id:3958578 N2 - 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. IS - 11 JF - The journal of gene medicine SN - 1521-2254 EP - 1269 TI - Minicircle-DNA production by site specific recombination and protein-DNA interaction chromatography. VL - 10 SP - 1253 KW - gene-therapy KW - plasmid AU - Mayrhofer, P AU - Blaesen, M AU - Schleef, M AU - Jechlinger, W PY - 2008/11// UR - http://dx.doi.org/10.1002/jgm.1243 ER -