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.
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.
