Antisense inhibitors for enhanced plasmid production

描述(申请人提供)：随着下一代生物技术产品(基因药物和DNA疫苗)进入临床试验，并最终进入制药市场，大规模生产质粒DNA正变得越来越重要。尽管对质粒DNA纯化方法的广泛研究已经导致了高效的纯化过程，但由于发酵产量低，质粒DNA的生产能力受到严重限制。该工作的假设是，通过靶向ColEI起始区抑制RNA(RNAi)的反义RNA的共表达，可以更有效地生产大肠杆菌质粒DNA。该策略将是制造设计的反义RNAi抑制剂，在适当的时间诱导它们，以允许目标质粒的失控复制。在特定的目标1中，我们将构建表达RNAi抑制物的重组载体。在特定的目标2中，我们将筛选对生产质粒有用的反义RNAi抑制剂。拟议的可行性研究的总体目标是确定这种细菌菌株在利用现有复制子生产质粒DNA方面是否具有显著优势(里程碑I)。如果成功，第二阶段的研究将包括：1)将反义RNA插入到大肠杆菌染色体中的改进结构；2)创造所需的菌株；3)发酵过程的开发。在第三阶段，NTC将把改进的菌株引入商业化的质粒生产。 潜在的商业应用 成功构建一个或多个以可控方式表达RNAi抑制物的有用细菌株(里程碑II)将有助于克服经济地生产用于人类制药和DNA疫苗的高纯度质粒DNA的主要障碍。这些菌株将被迅速引入自然技术公司的大规模DNA制造过程，并将通过该公司的技术转让和许可计划向商业用户提供许可。目前，实验性的基因药物和DNA疫苗通常是以克为单位制造的，主要用于动物安全性和有效性研究。然而，FDA批准的商业产品的出现将需要扩大到公斤，例如，广泛使用的疫苗。从目前生产的每克DNA 22,000-46,000美元的价格来看，迫切需要一种成本更低、可扩展的发酵过程。由此产生的产品的经济效益和潜在的市场规模可以很容易地得到评价。拟议的工作产生商业上有用和有价值的产品的可能性被认为很高。

DESCRIPTION (provided by applicant): Large scale manufacture of plasmid DNA is becoming increasingly important as the next generation of biotechnology products (gene medicines and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. Although extensive research on plasmid DNA purification methodologies has resulted in efficient purification processes, plasmid DNA production capacity is severely limited by low fermentation yield. The hypothesis of the proposed work is that E. coli plasmid DNA can be more efficiently produced by co-expression of an antisense RNA targeting the ColEI origin represser RNA (RNAI). The strategy will be to make designer antisense RNAI inhibitors, inducing them at the appropriate time to allow runaway replication of target plasmids. In Specific Aim 1, we will construct recombinant plasmids for expression of RNAI inhibitors. In Specific Aim 2, we will screen for antisense RNAI inhibitors useful for the production of plasmids. The overall goal of the proposed feasibility study is to determine whether such a bacterial strain is a significant advantage in the production of plasmid DNA using existing replicons (milestone I). If successful, Phase II studies will include: 1) improved constructs for insertion of the antisense RNA into the E. coli chromosome; 2) creating the strains needed and 3) fermentation process development. In Phase III, NTC will introduce the improved strains into commercial plasmid production. Potential commercial applications Success in constructing one or more useful strains of bacteria expressing RNAI inhibitor in a controllable fashion (milestone II) will help to overcome a major obstacle in the economical production of highly purified plasmid DNA for use in human Pharmaceuticals and DNA vaccines. Such strains will be rapidly introduced into Nature Technology's large scale DNA manufacturing process, and will also be made available for licensing to commercial users through the company's technology transfer and licensing program. Currently, experimental gene drugs and DNA vaccines are routinely made on the gram scale, mostly for use in animal safety and efficacy studies. However, the advent of FDA approved commercial products will require scaling up to kilograms, for example, for a widely used vaccine. Judging from the current price of $22,000- $46,000/gram of DNA produced, a less costly, scalable fermentation process is badly needed. The economic benefits and potential market size for the resulting products can be readily appreciated. The likelihood of a commercially useful and valuable product resulting from the proposed work is considered high.