Cobra Therapeutics Limited

David R Thatcher

Anthony Hitchcock

Diane L Varley

Roger Kingdon Craig

Robin E Offord

Keith Rose

Alistair Simpson Irvine

Julian A J Hanak

Adrian Haines

Julian Aj Hanak

Ross Owen Phillips

John Hamilton Welsh

Paula E Wilks

Hubert F Gaertner

A scalable method for the production of highly purified plasmid DNA in 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

The invention encompasses a transfection complex comprising a polypeptide comprising the contiguous 29 amino acids: 6G's, 2F's, 2L's, 1W, 4R's, 2E's, 2N's, 3K's, 1T, 1S, 1A, 1Y, 1M, 1C, and 1I, and having additional cationic residues to provide a net number of positive charges of equal to or greater than 8, and an isolated nucleic acid, and methods of transfecting cells using this transfection complex. The invention also includes a polypeptide having an amino acid composition including these 29 amino acids, as well as mixtures of polypeptides in which this polypeptide is present.

Compositions and methods for highly efficient transfection

Methods of screening for peptides useful in a gene delivery system to provide optimal transfection of cells based on kinetic parameters of the peptide-nucleic acid bimolecular interaction are described.

Optimization of gene delivery and gene delivery system

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.

The invention is based on the discovery of a pharmaceutical preparation for use in gene therapy which includes a therapeutic nucleic acid associated with Insulin-NHCO--CH.sub.2 --O--N.dbd.CH--C-Lys.sub.18 -Cys(S-Pyridyl)-OH in combination with a pharmaceutically acceptable carrier.

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Macclesfield, United Kingdom

David R Thatcher