Small interfering RNAs (siRNAs) and small hairpin RNAs (shRNAs) are potent gene-inhibiting agents that have been successfully used in cell culture. However, so far their application in vivo has been limited by insufficient delivery to target tissues. Because RNAs are susceptible to degradation by ribonucleases, particularly in the blood, a number of stabilizing chemical modifications have been tested for siRNAs. Some of these modifications have provided a dramatic increase of stability while maintaining high biological activity. However, there are no reports on the chemical modification of shRNA, and the choice and location of modifications is not obvious. This is because, unlike siRNAs, shRNA require enzymatic processing to cleave the terminal loop, and any modifications need to preserve that activity. While it is known that longer shRNAs are processed by enzyme Dicer, some results indicate that short shRNAs are processed by a distinct but unknown pathway. We have identified an unmodified shRNA that is a potent inhibitor of hepatitis C virus. We propose to search for stabilizing chemical modifications that will increase the shRNA half-life in blood while maintaining high biological activity. We propose to synthesize a set of shRNA molecules with various patterns of chemical modification, assay their stability in blood serum, then test their activity in a reporter system and in an HCV replicon system, both in human cells. The stabilized shRNAs that most successfully combine nuclease resistance with high potency will be used in an anti-HCV drug development program. s Hepatitis C virus (HCV) infects 175 million people worldwide, with 70% of patients developing chronic liver disease, including cirrhosis and hepatocellular carcinoma. There is no vaccine and current treatments are often ineffective and have severe side-effects. RNA interference (RNAi) is a newly emerging technology allowing potent inhibition of gene expression, with great potential for therapeutic use. shRNA are a promising class of RNAi drugs, but their use requires chemical stabilization to prevent their degradation in the body. This proposal will determine how to effectively accomplish this stabilization. [unreadable] [unreadable] [unreadable]