Both short interfering RNA (siRNA) and short hairpin RNA (shRNA) are potent gene-silencing agents that have been successfully used in cell culture. However, so far their application in vivo has been limited by the low stability of RNA and poor efficiency of delivery. The attachment of cholesterol groups to antisense oligonucleotides has been shown to improve their delivery, and it was recently demonstrated that cholesterol attached to the 5'-termini of the sense strands of siRNAs improve uptake both in cultured cells and in livers of living mice. In this proposal we develop methods to increase liver delivery and uptake by shRNAs. In contrast to siRNA, which directly goes to RISC complex, shRNA must be first processed by the enzyme Dicer to cleave the hairpin loop. Hence any chemical modifications must not block its ability to act as a substrate for Dicer. All modifications will be tested on a potent shRNA inhibitor of HCV discovered at SomaGenics for improved ability to inhibit HCV-mediated reporter expression both in a human hepatocyte cell line and in a mice liver model, assayed by whole-animal imaging. They will be also tested on a more realistic cell-based model of HCV, the HCV replicon, under SomaGenics' existing agreement with the NIAID antivirial screening program. [unreadable] [unreadable] [unreadable]