Amplification of Custom RNA in Plants

描述(申请人提供)：基于RNA的技术已经成为 自发现以来在研究和生物技术方面日益突出 催化RNA(核酶)，用于从 随机RNA文库，以及微阵列的广泛使用。然而， 通过合成或酶法生产RNA的成本仍然很高-至少10 是DNA成本的三倍，合成方法仅限于短RNA。这 Proposal描述了一种低成本、大规模生产的新方法 利用病毒在植物中扩增出任意长度的定制RNA产物 向量，这种方法已经成功地用于生产 蛋白质产品。在感染了重组病毒后，这些植物 收获后，使用一种既定的一步程序分离病毒粒子， RNA被提取，所需的片段在简单的 程序。病毒RNA的产量约占总干株的0.5% 质量。在这个项目的第一阶段，我们通过以下方式建立了原则证明 展示了将定制RNA片段插入到病毒RNA载体中， 在正常储存和体内保存下保持完全稳定和完整 条件。添加新的溶液组件后，插入物为 有效地从载体中去除。在第二阶段，我们将开发这种方法 变成了一种准备商业化的实用方法。这将涉及一系列 进行优化，使系统足够健壮，可以克服任何障碍 对于某些定制RNA来说，这可能会出现。我们的第一个大规模演示 从植物中产生的RNA将高达数百毫克 治疗性核酶和丙型肝炎病毒的关键成分。 建议的商业应用： RNA是结构生物学和包括HIV在内的生物医学研究的重要靶点 和流感病毒。它在反义和核酶治疗方面也有潜力， 生物技术产品和分子生物学工具：我们的技术将提供 与替代方案相比，大量定制RNA更便宜、更高效 方法：研究方法。我们目前的业务战略是与以下公司合作销售定制RNA 并将这项技术授权给某些其他制造商。

DESCRIPTION (provided by applicant): RNA-based technologies have become increasingly prominent in research and biotechnology since the discovery of catalytic RNA (ribozymes), techniques for selection of novel functions from random libraries of RNA, and the widespread use of microarrays. However, the cost of producing RNA by synthetic or enzymatic means remains high-at least ten times the cost of DNA, and synthetic methods are limited to short RNAs. This proposal describes a novel method for the low-cost, largescale production of custom RNA products of any length based on amplification in plants using viral vectors, an approach that has been successfully used for the production of protein products. After infection with recombinant virus, the plants are harvested, virions are isolated using an established, single-step procedure, RNA is extracted, and the desired fragment is excised and purified in a simple procedure. Yields of viral RNA are approximately 0.5 percent of total dry plant mass. In Phase I of this project we established proof of principle by demonstrating the insertion of a custom RNA fragment into the viral RNA vector, which remained completely stable and intact under normal storage and in vivo conditions. Upon addition of a new solution component, the insert was efficiently excised from the vector. In Phase II, we will develop this approach into a practical method ready for commercialization. This will involve a series of optimizations to make the system robust enough to overcome any obstacles that may emerge for certain custom RNAs. Our first demonstration of large-scale RNA production from plants will be to several hundred milligrams of a candidate therapeutic ribozyme and a key element of the hepatitis C virus. PROPOSED COMMERCIAL APPLICATION: RNA is an important target for structural biology and biomedical studied including HIV and influenza viruses. It also has potential in antisense and ribozyme therapeutics, biotechnology products and molecular biology tools: Our technology would provide large amounts of custom RNAs less expensively and more efficiently than alternative methods. Our current business strategy is to sell custom RNA in partnership with Large Scale Biology Corp. and license the technology to certain other manufacturers.