Cobra Therapeutics Limited

Julian Aj Hanak

Steven Geraint Williams

David R Thatcher

David J Sherratt

Anthony Hitchcock

Diane L Varley

A scalable method for the production of highly purified plasmid DNA in Escherichia coli is described, which method includes growing plasmid-containing cells to a high biomass in exponential growth and lysing the cells by raising the pH of the culture to a carefully controlled pH value in which chromosomal DNA is denatured but plasmid DNA is reversibly renatured. The method has been developed for the production of pharmaceutical grade DNA for use in in vivo and ex vivo gene therapy.

Method of plasmid DNA production and purification

A system is described which utilizes a novel system of repressor titration for maintenance of a plasmid useful in gene therapy and production of a recombinant protein. The system utilizes a transformed host cell containing a plasmid including an operator susceptible to binding by a repressor expressed in trans, a first chromosomal gene encoding the repressor, and a second chromosomal gene that is functionally associated with an operator and essential for cell growth, wherein the plasmid is present in the cell in sufficient numbers to titrate the repressor such that the essential gene is expressed, thereby permitting cell growth.

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Macclesfield, United Kingdom

Julian Aj Hanak