Cooke, JR; McKie, EA; Ward, JM; Keshavarz-Moore, E; (2004) Impact of intrinsic DNA structure on processing of plasmids for gene therapy and DNA vaccines. Journal of Biotechnology , 114 (3) 239 - 254. 10.1016/j.jbiotec.2004.06.011.
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Several non-Watson Crick DNA structures have been discovered to date, which may be incorporated into future plasmid constructs for gene therapy and DNA vaccine products. In this study, intrinsic DNA structures were included at a defined point in a 2.9 kb plasmid, and their effects on cell growth rate, total plasmid yield, and topology (i.e. the relative proportions of supercoiled plasmid, open circular and linear forms), were determined. The stability of the inserted sequences were assessed using gel electrophoresis. Z-DNA was shown to be unstable in a batch Escherichia coli DH1 production system grown in complex medium. Encouragingly other sequences studied (triplex, bend and quadruplex) did not cause spontaneous deletions, and no detrimental effect was found on growth rate or on total plasmid yield; indicating that such sequences could be included in future DNA products without any detrimental effect on plasmid yields; although the intra molecular triplex studied significantly decreased the proportion of supercoiled species.
|Title:||Impact of intrinsic DNA structure on processing of plasmids for gene therapy and DNA vaccines|
|Additional information:||Sequences taken from viruses for DNA vaccines, and from the human genome for future gene therapy products, may contain elements that produce unusual secondary DNA structures. These may result in adverse effects on the production, stability and ultimately safety of the product. This study determined the implications such unusual structural features have on plasmid production. The results have been used considered in the design of novel plasmid based therapies.|
|Keywords:||DNA vaccines, Gene Therapy, Plasmid production, Triplex-DNA, Plasmid stability, Z-DNA|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology|
UCL > School of BEAMS > Faculty of Engineering Science > Biochemical Engineering
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