Antisense inhibitors for enhanced plasmid production

用于增强质粒生产的反义抑制剂

基本信息

批准号：
6883528
负责人：
James Williams
金额：
$ 13.62万
依托单位：
NATURE TECHNOLOGY CORPORATION
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-06-01 至 2006-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6883528
关键词：
Escherichia coli RNA interference antisense nucleic acid bacterial genetics biotechnology fermentation gene expression genetic recombination genetic strain molecular cloning nucleic acid chemical synthesis plasmids replicon small interfering RNA technology /technique development transfection

项目摘要

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.

描述（由申请人提供）：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，最终进入药品市场，质粒DNA的大规模生产变得越来越重要。尽管对质粒DNA纯化方法的广泛研究导致了有效的纯化过程，但质粒DNA产量受到低发酵产量受到严重限制。提出的工作的假设是，大肠杆菌质粒DNA可以通过共表达Colei起源抑制剂RNA（RNAI）的反义RNA来更有效地产生。该策略将是使设计师反义RNAi抑制剂在适当的时间诱导它们，以使靶质粒复制失控。在特定的目标1中，我们将构建重组质粒以表达RNAi抑制剂。在特定的目标2中，我们将筛选对质粒生产有用的反义RNAi抑制剂。拟议的可行性研究的总体目标是确定使用现有复制子（Milestone I）在产生质粒DNA的重要优势。如果成功，第二阶段的研究将包括：1）改进的构建体以将反义RNA插入大肠杆菌染色体中； 2）创建所需的菌株和3）发酵过程开发。在第三阶段，NTC将将改善的菌株引入商业质粒生产中。潜在的商业应用成功地以可控的方式构建一种或多种有用的细菌菌株（Milestone II）将有助于克服经济生产高度纯化的质粒DNA的主要障碍，用于人类药物和DNA疫苗。此类菌株将迅速引入自然技术的大规模DNA制造过程中，并通过公司的技术转移和许可计划也可以向商业用户提供许可。目前，实验性基因药物和DNA疫苗通常是按照量表进行的，主要用于动物安全和功效研究。但是，FDA批准的商业产品的出现将需要扩展多达公斤，例如，使用广泛使用的疫苗。从目前的价格为$ 22,000- $ 46,000/克的DNA的价格来看，急需成本较低，可扩展的发酵过程。可以很容易地赞赏所得产品的经济利益和潜在的市场规模。拟议的工作产生的商业有用和有价值的产品的可能性被认为很高。