Improved bacterial strains for therapeutic DNA and protein production

用于治疗性 DNA 和蛋白质生产的改良菌株

• 批准号: 7929485
• 负责人: FREDERICK R BLATTNER
• 金额: $ 83.68万
• 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 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7929485
• 关键词: Bacteria; Bacteriophages; Biologic Development; Biotechnology; Cellular Structures; Chromosomes, Human, Pair 20; Clinical Trials; Code; Collaborations; Complex; Contracts; DNA; Disease; Drug Industry; Endotoxins; Escherichia coli; FDA approved; Fermentation; Gene Deletion; Gene Mutation; Gene Targeting; Generations; Genes; Genetic Transcription; Genome; Genomics; Goals; Grant; Growth; Health; Human; Inflammation; Insulin; Lead; Lipids; Lipopolysaccharide Biosynthesis Pathway; Lipopolysaccharides; Mammals; Manufacturer Name; Marketing; Membrane; Metabolic; Methods; Mus; Nucleic Acids; Peptide Synthesis; Perception; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Plasmids; Polymerase; Preparation; Production; Proteins; Research; Risk; Safety; Sampling; Side; Structure; System; Technology; Testing; Therapeutic; Toxic effect; Toxin; Transfection; Transposase; Vaccines; Virulent; bacterial vector; base; commercial application; cost; drug isolation; gene therapy; genetic element; immune activation; improved; in vivo; innovation; large scale production; mortality; mouse model; periplasm; plasmid DNA; public health relevance; sugar; therapeutic protein

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.

描述(由申请人提供)：这项第二阶段计划的目标是在“真实世界”的条件下评估Scarab基因组公司改进的大肠杆菌生产菌株，在这种条件下可以生产药理级别的治疗蛋白和质粒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Scarab的Clean Genome(R)大肠杆菌菌株制造的药物。 自1982年FDA批准大肠杆菌生产的人胰岛素上市以来，大肠杆菌发酵一直是商业化生产治疗性DNA和蛋白质的首选和成本效益高的方法。人们对大肠杆菌的表达系统进行了许多创新，但自20世纪70年代S以来，商业上使用的实际细菌并没有得到根本的改进，直到斯卡拉布的无疤痕基因组缺失技术使系统地修改大肠杆菌基因组成为可能。 这项技术使Scarab能够创造出缺乏许多不利于商业应用的基因(高达20%的染色体)的“减少基因组”的大肠杆菌菌株。“清洁基因组”菌株提高了代谢效率和产品产量，增加了克隆稳定性，并消除了许多不必要的发酵副产物，包括噬菌体、鞭毛、菌毛、转座酶，在某些情况下还消除了毒素。 该项目的第一阶段涉及限制最严重的大肠杆菌毒素--内毒素(也称为脂多糖或脂多糖)的问题，目前所有使用的大肠杆菌生产菌株中都存在这种毒素。FDA对药品中的内毒素水平进行了严格的限制。内毒素无法完全消除，因为编码核心内毒素结构的基因对生长至关重要，但在第一阶段所做的发现导致了基因突变，大大降低了内毒素水平。我们最好的低内毒素大肠杆菌，当注射到小鼠体内时，死亡率比目前生产中使用的普通大肠杆菌降低了10倍以上。 希望这个第二阶段的项目将导致FDA申请临床试验至少一种由我们的合作者之一的Scarab菌株生产的药物。证明Scarab的菌株已获得FDA批准，将消除我们营销的一个重大障碍，即保守的看法，即以前批准的就足够好了。 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，因为样品可能会受到包括内毒素在内的杂质的污染。由于含有内毒素等杂质的治疗药物具有健康风险，而且提纯成本较高，因此产生减少杂质携带和毒性的细菌菌株对生物和制药行业具有巨大的价值。