Chimeric enzyme for nucleic acid autohydrolysis

描述（由申请人提供）：从细菌质粒中提取的DNA疫苗和基因药物正在成为一类重要的新型药物。严格按照合理设计制造的DNA分子，使制造商无需花费数年时间研制有效疫苗，只需几天就能研制出新的疫苗实体，并在2-3周内批量生产疫苗，以便快速部署对抗新的生物制剂。然而，现有的发酵和纯化生产技术效率低下，难以规模化，并且需要复杂的生产设备和方法。这限制了它们在满足现有质粒应用的成本和容量需求，或快速生产用于大流行疫苗接种的数公斤质粒DNA方面的效用。第一阶段直接成功地解决了这些问题，证明了一种成本效益显著的简化和简单的净化过程的可行性，消除了专门和昂贵的碱性或热分解步骤以及相关的有毒废物流。在第二阶段，我们将制作和评估设计菌株以及相关的发酵和纯化方法。这些菌株将利用在第一阶段开发的新型嵌合核酸酶和自溶细菌菌株。原型I是一种快速部署质粒生产系统，将自溶质粒纯化与自然技术公司（NTC）现有的发酵平台连接起来，以促进立即生产用于大流行应用的各种质粒dna。该平台的主要驱动因素是生产速度、安全性、简单性、可扩展性以及可转移到发达国家和新兴国家现有生产设施的能力。将创建具有集成嵌合核酸酶的自溶菌株，并开发发酵，自溶和质粒纯化方法。该系统将被整合到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旨在促进基因药物的商业化，用于各种应用。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）、可扩展性和安全性。原型IV链接高产确定的培养基发酵与新的简化的裂解和纯化方法。这项技术将授权给质粒DNA制造商；已经确定了一个这样的商业化渠道。

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.