Improved bacterial strains for therapeutic DNA and protein production

描述（由申请人提供）：本阶段II提案的目标是评估圣甲虫基因组公司的改进E。大肠杆菌生产菌株在“真实的世界”的条件下，在该条件下产生药理学级治疗性蛋白质和质粒DNA。在某些情况下，这将与使用GMP设施的合同制造商合作完成。候选蛋白质和质粒将通过与生物技术公司的合作获得，这些公司正在开发可以用圣甲虫的清洁基因组（r）E生产的药物。大肠杆菌菌株。 自从1982年FDA批准由E.大肠杆菌、大肠埃希菌等。大肠杆菌发酵已经成为商业生产治疗性DNA和蛋白质的优选和成本有效的方法。在表达系统方面，人们对E.大肠杆菌的基因组，但实际的商业使用的细菌从20世纪70年代以来一直没有得到基本的改进，直到圣甲虫的无瘢痕基因组删除技术使得系统地修改大肠杆菌的基因组成为可能。杆菌 这项技术使圣甲虫能够创造出“简化基因组”的E。大肠杆菌缺乏许多不利于商业应用的基因（高达20%的染色体）。“清洁基因组”菌株具有改善的代谢效率和产物产率，增加的克隆稳定性，并且消除了许多不期望的发酵副产物，包括噬菌体、鞭毛、菌毛、转座酶和在某些情况下的毒素。 该项目的第一阶段处理限制最严重的大肠杆菌毒素，内毒素（也称为脂多糖或LPS）的问题，内毒素在所有目前使用的大肠杆菌中都以最大强度存在。大肠杆菌生产菌株。FDA对药品中的内毒素水平有严格的限制。内毒素不能完全消除，因为编码核心LPS结构的基因对生长至关重要，但在I期的发现导致基因突变，大大降低了内毒素的水平。我们最好的低内毒素E。大肠杆菌注射到小鼠体内后，死亡率比普通大肠杆菌降低了10倍以上。目前生产中使用的。 我们希望这个II期项目将导致FDA申请对我们的合作者之一的圣甲虫菌株生产的至少一种药品进行临床试验。圣甲虫菌株已被FDA批准的证明将消除我们营销的一个重大障碍，即保守的看法，即以前批准的已经足够好了。 公共卫生相关性：细菌发酵产生的质粒DNA和蛋白质受到严格的纯度限制，因为样品可能被包括内毒素在内的杂质污染。由于含有内毒素等杂质的治疗剂的健康风险以及由于纯化的高成本，产生减少杂质携带和毒性的细菌菌株对生物和制药工业具有巨大价值。

DESCRIPTION (provided by applicant): The goal of this Phase II proposal is to evaluate Scarab Genomics' improved E. coli production strains in "real world" conditions under which pharmacological grade therapeutic proteins and plasmid DNA are produced. In some cases this will be done in collaboration with contract manufacturers using GMP facilities. Candidate proteins and plasmids will be obtained through collaborations with biotech companies which are developing pharmaceuticals that could be manufactured with Scarab's Clean Genome(r) E. coli strains. Since 1982, when the FDA approved human insulin produced by E. coli for marketing, E. coli fermentation has been a preferred and cost-effective method for commercial production of therapeutic DNA and proteins. Many innovations have been made to the expression systems used with E. coli, but the actual bacteria used commercially had not been basically improved since the 1970's until Scarab's scarless genomic deletion technology made it possible to systematically revise the genome of E. coli. This technology has enabled Scarab to create "reduced genome" strains of E. coli that lack many genes (up to 20% of the chromosome) that are not beneficial for commercial applications. The "clean genome" strains have improved metabolic efficiency and product yield, increased clone stability and have eliminated many undesired fermentation side products including phage, flagellae, fimbrae, transposases and in some cases, toxins. Phase I of this project dealt with the problem of limiting the most serious E.coli toxin, endotoxin (also called lipopolysacharide or LPS), which is present at full strength in all currently used E. coli production strains. The FDA places strict limits on endotoxin levels in pharmaceuticals. Endotoxin cannot be completely eliminated because the genes coding for the core LPS structure are essential for growth, but discoveries made in Phase I led to genetic mutations reducing the level of endotoxin substantially. The best of our low endotoxin E. coli, when injected into mice, decreases mortality by more than 10-fold over the ordinary E. coli currently used in production. It is hoped that this Phase II project will lead to an FDA application for clinical trial of at least one pharmaceutical product produced by a Scarab strain from one of our collaborators. A demonstration that Scarab's strain has been FDA approved will remove a significant barrier to our marketing, namely the conservative perception that what has been approved before is good enough. PUBLIC HEALTH RELEVANCE: Plasmid DNA and protein generated from bacterial fermentation is subject to stringent purity constraints because of the potential for contamination of samples with impurities that include endotoxin. Because of the health risk of therapeutics that contain impurities like endotoxin and due to the high cost of purification, generating bacterial strains that reduce impurity carryover and toxicity are of tremendous value to the biologic and pharmaceutical industry.