STTR Phase I: Scaleable, Inexpensive Production of siRNA in E. coli

这个小企业技术转移第一阶段项目开发分子生物学、生化工程和纯化技术，以提供与现有技术相比规模较大、成本较低的“小干扰RNA”或siRNA分子。近年来，通过“RNA干扰”(RNAi)，siRNA被认为是一种有效的基因沉默试剂。癌症、艾滋病毒和肝炎等重要疾病的基因已经被证明可以通过siRNA作用进行控制。SiRNA的应用现在保证了大规模制造siRNA用于RNAi相关研究和作为药物的目的。这项技术的独特之处在于我们得到了充分的证据，证明RNA插入序列可以保存在更大的核糖体RNA(RRNA)载体中，并通过常规发酵在大肠杆菌中高水平生产。在发酵后，提取和纯化所需的siRNA以用于大规模生产RNA产品。本研究的更广泛的影响是提供一种通用的大规模生产工具，适用于未来广泛的新型RNA产品。除了siRNA的生产外，这里提出的方法还可以用于大规模生产微小RNA、适体、反义疗法或其他短RNA分子，这些分子在结构和药理学应用中也具有相当大的兴趣。相关的产品和RNA表达系统预计将具有相当大的商业价值，首先是在研究界，最终是作为药物。该项目还将导致有关RNA结构、酶作用和特定RNA分子的纯化的基础科学知识。

This Small Business Technology Transfer Phase I project develops molecular biology, biochemical engineering, and purification techniques to provide "small interfering RNA" or siRNA molecules on at large-scale and at low cost compared to available techniques. In recent years, siRNA's have been recognized as potent gene-silencing agents via "RNA interference" (RNAi). Genes for important diseases such as cancer, HIV, and hepatitis have already been shown to be controllable by siRNA action. Applications of siRNA's now warrant large-scale manufacture of siRNAs for the purposes of RNAi associated studies and as pharmaceutical agents. Unique to the technology is our well demonstrated evidence that RNA insert sequences can be harbored in larger ribosomal RNA (rRNA) carriers and produced at high levels within E. coli by conventional fermentation. Following fermentation, the desired siRNA is excised and purified for large-scale production of the RNA product.The broader impacts of this research is to provide a general, large-scale production tool applicable to a wide range of future novel RNA products. In addition to siRNA production, the methods proposed here can be used for large-scale production of micro-RNAs, aptamers, antisense therapeutics, or other short RNA molecules that are also of considerable interest for structural and pharmacological applications. The associated products and RNA expression systems are expected to have considerable commercial value, first in the research community and eventually as pharmaceuticals. This project will also result in fundamental scientific knowledge regarding RNA structure, enzymatic action, and purification of particular RNA molecules.