Bacterial Lipopolysaccharide Copurifies with Plasmid DNA: Implications for Animal Models and Human Gene Therapy

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Abstract
During the course of gene therapy experiments in rodents, using intramuscular injections of plasmid DNA derived from Escherichia coli, we noted dose-related toxicity. This observation prompted a search for possible contaminants of DNA samples. We used the highly specific and sensitive limulus amoebocyte lysate assay (LAL), to monitor endotoxin bioactivity in DNA samples, and found plasmid DNA derived from standard E. coli 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 to 500 μg/ml of LPS. LPS is a potent inducer of cytokines and other inflammatory mediators, and may complicate the use of naked DNA in gene therapy. The copurification of endotoxin with plasmid DNA also has important implications for in vitro transfection studies and microinjection 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, incubation with lysozyme, treatment with the detergent n-octyl-β-d-thioglucopyranoside (OSPG) and polymyxin-B (PMB) chromatography, allowed the isolation of plasmid DNA containing less than 50 ng/ml LPS. This represents a 10,000-fold reduction in LPS contamination, compared to conventional methods of plasmid DNA purification, avoids potentially toxic reagents such as ethidium bromide, and produces a higher yield of plasmid DNA. Several recent experiments in gene therapy have focused on the modulation of immune responses. Bacterial lipopolysaccharide (LPS) is a potent lymphocyte mitogen and adjuvant that can confound the results of gene immunotherapy studies. Plasmid DNA prepared from E. coli by standard techniques is highly contaminated with LPS. We describe here a simple method to remove LPS from DNA by the conversion of bacteria to spheroplasts, followed by treatment with a nonionic detergent and polymyxin B chromatography. DNA prepared by this method is essentially free of LPS, and, unlike conventionally purified plasmid DNA, does not produce nonspecific immune activation following in vivo administration.