Improved bacterial strains for therapeutic DNA production

点击翻译按钮获取中文摘要

The goal of this proposal is to develop methods and strains for manufacturing plasmid DNA of extraordinary purity in very large quantity for therapeutic use. Specifically we propose to use genetic techniques to lower the level of contaminating endotoxin in plasmid DNA preparations by many orders of magnitude. The advent of DNA based vaccines, gene therapy approaches and plasmid based RNA interference (RNAi) has opened the way for DNA to be used directly as a therapeutic or preventative agent against viral or other infectious diseases, some forms of cancer and possibly to ameliorate inborn genetic diseases. This has created a market for large quantities of injection grade plasmid DNA. For clinical trials, and ultimately administration to patients, DNA preparations must be manufactured to the highest specifications of quality and safety. Removal of endotoxin is critical to achievement of satisfactory purity. E. coli K12 has been used for decades to produce plasmid DNA for molecular biology research and this methodology has in general simply been extended to manufacturing practice. A critical problem with the use of E. coli, however, is the carryover of contaminants from the host into the finished product. Potential contaminants include host proteins, transposable elements that can jump from the host into the product plasmid DNA and highly toxic lipopolysaccharide moieties from the outer membrane of the bacteria collectively known as endotoxin. Scarab Genomics has developed and patented its reduced genome E. coli strains which remove the genes for 650 potentially contaminating proteins and all transposable elements from the chromosome. Endotoxin, has until now been difficult to remove because it is essential to the E. coli cell. Moreover endotoxin is in reality a heterogeneous mixture so a single purification step is not 100% effective. Success in this project will come from genomic simplification of endotoxin to a single uniform species which can be completely removed by a single simple chromatography step. These new Clean Genome E. coli strains will be of great medical benefit in providing large quantities of safer DNA at low cost for therapeutic use.