BACTERIAL LIPOPOLYSACCHARIDE COPURIFIES WITH PLASMID DNA - IMPLICATIONS FOR ANIMAL-MODELS AND HUMAN GENE-THERAPY

During the course of gene therapy experiments in rodents, using intram uscular injections of plasmid DNA derived from Escherichia coil, we no ted dose-related toxicity. This observation prompted a search for poss ible contaminants of DNA samples. We used the highly specific and sens itive limulus amoebocyte lysate assay (LAL), to monitor endotoxin bioa ctivity in DNA samples, and found plasmid DNA derived from standard E. coil bacterial strains, using traditional DNA isolation protocols, to be heavily contaminated with endotoxin, or lipopolysaccharide (LPA). Standard DNA isolation procedures result in the copurification of up t o 500 mu g/ml of LPS. LPS is a potent inducer of cytokines and other i nflammatory mediators, and may complicate the use of naked DNA in gene therapy. The copurification of endotoxin with plasmid DNA also has im portant implications for in vitro transfection studies and microinject ion of DNA into embryos. A simple and efficient protocol to reduce LPS contamination of plasmid DNA was developed. The conversion of intact bacteria to spheroplasts prior to the isolation of plasmid DNA, incuba tion with lysozyme, treatment with the detergent n-octyl-beta-D-thiogl ucopyranoside (OSPG) and polymyxin-B (PMB) chromatography, allowed the isolation of plasmid DNA containing less than 50 ng/ml LPS. This repr esents a 10,000-fold reduction in LPS contamination, compared to conve ntional methods of plasmid DNA purification, avoids potentially toxic reagents such as ethidium bromide, and produces a higher yield of plas mid DNA.