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Bacterial lipopolysaccharide copurifies with plasmid DNA: implications for animal models and human gene therapy
I P Wicks1, M L Howell, T Hancock
1Department of Medicine, University of California, San Diego, La Jolla 92093-0663, USA.
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 resulted in the copurification of up to 500 micrograms/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-beta-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.
Insights
Gene therapy using plasmid DNA faces challenges due to endotoxin (lipopolysaccharide, LPS) contamination. A new protocol significantly reduces LPS levels, improving DNA safety for therapeutic applications.
Area of Science:
- Molecular Biology
- Gene Therapy
- Biochemistry
Background:
- Plasmid DNA is crucial for gene therapy but can be contaminated with endotoxins (lipopolysaccharide, LPS) from bacterial sources.
- Endotoxin contamination in plasmid DNA can cause dose-related toxicity and inflammatory responses, complicating therapeutic use and research.
- Traditional DNA isolation methods often fail to remove significant amounts of LPS, leading to high contamination levels (up to 500 µg/ml).
Purpose of the Study:
- To investigate endotoxin contamination in plasmid DNA prepared using standard protocols.
- To develop an efficient method for reducing lipopolysaccharide (LPS) contamination in plasmid DNA.
- To assess the impact of LPS on gene therapy applications and in vitro studies.
Main Methods:
- Utilized the Limulus amoebocyte lysate (LAL) assay to quantify endotoxin (LPS) levels in plasmid DNA samples.
- Developed a novel purification protocol involving bacterial spheroplast formation, lysozyme treatment, and polymyxin-B chromatography.
- Compared LPS levels in DNA purified by the new method versus conventional techniques.
Main Results:
- Standard plasmid DNA isolation yielded high levels of LPS (up to 500 µg/ml).
- The developed protocol reduced LPS contamination to less than 50 ng/ml, a 10,000-fold decrease.
- The new method avoids toxic reagents like ethidium bromide and increases plasmid DNA yield.
Conclusions:
- Plasmid DNA derived from standard bacterial strains is significantly contaminated with LPS using conventional purification.
- The novel protocol effectively removes LPS, producing highly purified plasmid DNA suitable for gene therapy and other applications.
- Reducing endotoxin contamination is critical for the safe and effective use of naked DNA in molecular biology and medicine.