Amplification of Custom RNA in Plants

描述（由申请人提供）：基于RNA的技术已成为 在研究和生物技术中日益突出，因为发现了 催化RNA（核酶），用于选择新功能的技术， RNA的随机文库，以及微阵列的广泛使用。但 通过合成或酶促方法生产RNA的成本仍然很高--至少10 是DNA成本的两倍，合成方法仅限于短RNA。这 该提案描述了一种低成本，大规模生产的新方法， 基于使用病毒在植物中扩增的任何长度的定制RNA产物 载体，这种方法已成功地用于生产 蛋白质产品在用重组病毒感染后， 收获的病毒体使用已建立的一步法分离， 提取RNA，切除所需片段并在简单的酶切中纯化。 procedure.病毒RNA的产量约为总干植物的0.5% 马萨诸塞州在该项目的第一阶段，我们通过以下方式建立了原理证明： 证明了定制RNA片段插入病毒RNA载体， 其在正常储存和体内保持完全稳定和完整 条件在添加新的溶液组分后， 从载体中有效地切除。在第二阶段，我们将发展这种方法， 转化为一种实用的方法，准备商业化。这将涉及一系列 使系统足够强大，以克服任何障碍 可能会出现特定的定制RNA。我们第一次大规模的 从植物中产生的RNA将是几百毫克的一种候选物 治疗性核酶和丙型肝炎病毒的关键元件。 拟定商业应用： RNA是结构生物学和包括HIV在内的生物医学研究的重要靶标 和流感病毒。它在反义和核酶治疗中也有潜力， 生物技术产品和分子生物学工具：我们的技术将提供 大量定制RNA比替代品更便宜且更有效 方法.我们目前的业务战略是与以下公司合作销售定制RNA： 大规模生物公司和许可证的技术，以某些其他制造商。

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.