Improved bacterial strains for therapeutic DNA production

本提案的目的是开发用于大量生产用于治疗用途的极高纯度的质粒DNA的方法和菌株。具体来说，我们建议使用遗传技术将质粒DNA制剂中污染内毒素的水平降低许多数量级。基于DNA的疫苗、基因治疗方法和基于质粒的RNA干扰（RNAi）的出现为DNA直接用作针对病毒或其他感染性疾病、某些形式的癌症的治疗剂或预防剂以及可能改善先天性遗传疾病开辟了道路。这为大量注射级质粒DNA创造了市场。对于临床试验，并最终给病人用药，DNA制剂必须按照最高的质量和安全规格生产。去除内毒素对于获得令人满意的纯度至关重要。E.大肠杆菌K12几十年来一直被用来生产质粒DNA用于分子生物学研究，这种方法通常只是简单地扩展到生产实践中。使用E.然而，大肠杆菌是污染物从宿主携带到成品中。潜在的污染物包括宿主蛋白质、可从宿主跳跃到产物质粒DNA中的转座因子和来自细菌外膜的高毒性脂多糖部分，统称为内毒素。圣甲虫基因组公司已经开发出其简化基因组E。大肠杆菌菌株，从染色体上去除了650种潜在污染蛋白质和所有转座因子的基因。内毒素是大肠杆菌所必需的，至今难以去除。coli细胞。此外，内毒素实际上是异质混合物，因此单个纯化步骤不是100%有效的。该项目的成功将来自于内毒素的基因组简化为单一的均匀物质，其可以通过单一的简单色谱步骤完全去除。这些新的清洁基因组E。大肠杆菌菌株将在以低成本提供大量更安全的DNA用于治疗用途方面具有巨大的医疗益处。

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.