Improved bacterial strains for therapeutic DNA production

用于治疗性 DNA 生产的改良菌株

• 批准号: 7271734
• 负责人: FREDERICK R BLATTNER
• 金额: $ 13.92万
• 依托单位: SCARAB GENOMICS, LLC
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2007-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7271734
• 关键词: Achievement; Acylation; Antigens; Bacteria; Bacteriophages; Biological Assay; Biological Products; Cell Survival; Cells; Characteristics; Chromatography; Chromosomes; Clinical; Clinical Trials; Communicable Diseases; DNA; DNA Transposable Elements; Endotoxins; Engineering; Enteral; Escherichia coli; Escherichia coli K12; Essential Genes; Excision; Fermentation; Gene Cluster; Genes; Genetic Techniques; Genome; Genomics; Goals; Inborn Genetic Diseases; Injection of therapeutic agent; Legal patent; Lipopolysaccharides; Malignant Neoplasms; Marketing; Medical; Membrane; Methods; Molecular Biology; Numbers; Patients; Plasmids; Preparation; Principal Investigator; Process; Production; Proteins; RNA Interference; Research Methodology; Resistance; Safety; Source; Staging; Structure; Therapeutic; Therapeutic Uses; Vaccines; Viral; abstracting; base; cost; gene therapy; improved; plasmid DNA; programs; success

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.

本提案的目标是开发方法和菌株，以制造非常高纯度的质粒DNA，用于大量治疗。具体而言，我们建议使用遗传技术将质粒DNA制备中的内毒素污染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直接用作治疗或预防病毒或其他传染病、某些形式的癌症以及可能改善先天遗传疾病开辟了道路。这为大量注射级质粒DNA创造了市场。为了临床试验和最终给病人用药，DNA制剂必须按照最高的质量和安全标准生产。去除内毒素是获得满意纯度的关键。大肠杆菌K12已经使用了几十年来生产质粒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。然而，使用大肠杆菌的一个关键问题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，可以从宿主跳到产物质粒DNA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。Scarab Genomics已经开发并申请了其减少基因组的大肠杆菌菌株的专利，该菌株可以从染色体上去除650种潜在污染蛋白质和所有转座因子的基因。到目前为止，内毒素很难去除，因为它对大肠杆菌细胞至关重要。此外，内毒素实际上是一种异质混合物，因此单一的纯化步骤不是100%有效的。这个项目的成功将来自于内毒素的基因组简化到一个单一的统一的物种，可以通过一个简单的色谱步骤完全去除。这些新的清洁基因组大肠杆菌菌株将具有巨大的医学效益，可以以低成本提供大量更安全的DNA用于治疗用途。