Chimeric enzyme for nucleic acid autohydrolysis

用于核酸自水解的嵌合酶

• 批准号: 7282957
• 负责人: James Williams
• 金额: $ 41.33万
• 依托单位: NATURE TECHNOLOGY CORPORATION
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-23 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7282957
• 关键词: Address; Animals; Autolysis; Bacteriophages; Biological Products; Biomass; Bypass; Cells; Class; Classification; Compatible; Country; Cytolysis; DNA; DNA Vaccines; Development; Endoribonucleases; Enzymes; Equipment; Escherichia coli; Evaluation; Excision; Exonuclease; Fermentation; Gene Transduction Agent; Genes; Genomics; Heating; Influenza; Influenza A Virus, H5N1 Subtype; Kilogram; Licensing; Link; Manufacturer Name; Medicine; Membrane; Methodology; Nature; Nucleic Acids; Pancreatic ribonuclease; Pharmacologic Substance; Phase; Plasmids; Process; Production; Proteins; RNA; Range; Reagent; Residual state; Ribonucleases; Safety; Speed; Stream; System; Technology; Testing; Time; Vaccination; Vaccines; Week; commercialization; concept; cost; cost effective; day; design; endolysin; gene replacement; immunogenicity; improved; manufacturing facility; novel; novel vaccines; nuclease; pandemic disease; periplasm; plasmid DNA; prototype; response; therapeutic gene; wasting

DESCRIPTION (provided by applicant): DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. DNA molecules made strictly by rational design allows the manufacturer to bypass years of development for the production of efficacious vaccines, and literally create new vaccine entities in days and mass produce vaccines in 2-3 weeks for rapid deployment against new biological agents. However, existing fermentation and purification production technologies are inefficient, difficult to scale, and require sophisticated production equipment and methodology. This limits their utility to meet cost and capacity needs for existing plasmid applications, or to rapidly produce kilograms of plasmid DNA for pandemic vaccination. Phase I addressed these problems directly and successfully by proving feasibility of a cost effective dramatically streamlined and simple purification process that eliminates specialized and costly alkaline or heat lysis steps and the associated toxic waste streams. In Phase II, we will make and evaluate designer strains and associated fermentation and purification methodology. These strains will utilize novel chimeric nucleases and autolytic bacterial strains developed in Phase I. Four prototypes are envisioned. Prototype I is a rapid deployment plasmid production system linking autolytic plasmid purification to Nature Technology Corporation's (NTC) existing fermentation platform to facilitate immediate production of a variety of plasmid DNAs for pandemic applications. The key drivers for this platform are speed of production, safety, simplicity, scalability and transferability to existing manufacturing facilities in developed and emerging countries. Autolytic strains with an integrated chimeric nuclease will be created, and fermentation, autolysis, and plasmid purification methodologies developed. The system will be integrated into NTC's existing rapid deployment DNA vaccine platform (RapidVACC) for response to influenza H5N1 pandemic. Prototypes II-IV are aimed at facilitating the commercialization of gene medicines for a variety of applications. The key drivers for these prototypes are purity, yield, cost (reagents, equipment, volumes, and time), scalability and safety. Prototype IV links high yield defined media fermentation with new simplified lysis and purification methodologies. This technology will be licensed to plasmid DNA manufacturers; one such commercialization channel has already been identified.

描述(申请人提供)：DNA疫苗和基因药物，来源于细菌质粒，正在成为一类重要的新药物。严格按照合理设计制造的DNA分子允许制造商绕过数年的开发来生产有效的疫苗，真正的在几天内创造出新的疫苗实体，并在2-3周内大规模生产疫苗，以针对新的生物制剂进行快速部署。然而，现有的发酵和纯化生产技术效率低，难以规模化，需要复杂的生产设备和方法。这限制了它们的实用性，不能满足现有质粒应用的成本和容量需求，也不能快速生产用于大流行疫苗接种的千克质粒DNA。第一阶段直接和成功地解决了这些问题，证明了一种成本效益高、大大简化和简单的净化工艺的可行性，该工艺消除了专门和昂贵的碱或热分解步骤和相关的有毒废流。在第二阶段，我们将制作和评估设计菌株和相关的发酵和纯化方法。这些菌株将利用第一阶段开发的新型嵌合核酸酶和自溶细菌菌株，预计有四个原型。Prototype I是一种快速部署的质粒生产系统，将自溶的质粒纯化与自然技术公司(NTC)现有的发酵平台连接起来，以便于立即生产各种用于大流行应用的质粒DNA。这一平台的关键驱动因素是生产速度、安全性、简单性、可扩展性以及对发达国家和新兴国家现有制造设施的可转移性。将创建具有整合嵌合核酸酶的自溶菌株，并开发发酵、自溶和质粒纯化方法。该系统将整合到NTC现有的快速部署DNA疫苗平台(RapidVACC)中，以应对H5N1流感大流行。原型II-IV旨在促进各种用途的基因药物的商业化。这些原型的关键驱动因素是纯度、产量、成本(试剂、设备、体积和时间)、可伸缩性和安全性。Prototype IV将高产量定义的介质发酵与新的简化裂解和纯化方法联系起来。这项技术将被授权给质粒DNA制造商；已经确定了一个这样的商业化渠道。

项目成果

Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields.

• DOI: 10.1002/jgm.1499
• 发表时间: 2010-10
• 期刊: JOURNAL OF GENE MEDICINE
• 影响因子: 3.5
• 作者: Carnes, Aaron E.;Luke, Jeremy M.;Vincent, Justin M.;Anderson, Sheryl;Schukar, Angela;Hodgson, Clague P.;Williams, James A.
• 通讯作者: Williams, James A.

Generic plasmid DNA production platform incorporating low metabolic burden seed-stock and fed-batch fermentation processes.

• DOI: 10.1002/bit.22347
• 发表时间: 2009-08-15
• 期刊: BIOTECHNOLOGY AND BIOENGINEERING
• 影响因子: 3.8
• 作者: Williams, James A.;Luke, Jeremy;Langtry, Sarah;Anderson, Sheryl;Hodgson, Clague P.;Carnes, Aaron E.
• 通讯作者: Carnes, Aaron E.

Improved antibiotic-free plasmid vector design by incorporation of transient expression enhancers.

通过掺入瞬态表达增强子，改善了无抗生素质粒载体设计。

• DOI: 10.1038/gt.2010.149
• 发表时间: 2011-04
• 期刊: Gene therapy
• 影响因子: 5.1
• 作者: Luke JM;Vincent JM;Du SX;Gerdemann U;Leen AM;Whalen RG;Hodgson CP;Williams JA
• 通讯作者: Williams JA