Search

RNA interference offers enormous potential to develop therapeutic agents for a variety of diseases. To assess the stability of siRNAs under conditions relevant to clinical use with particular emphasis on topical delivery considerations, a study of three different unmodified siRNAs was performed. The results indicate that neither repeated freeze/thaw cycles, extended incubations (over 1 year at 21 degrees C), nor shorter incubations at high temperatures (up to 95 degrees C) have any effect on siRNA integrity as measured by nondenaturing polyacrylamide gel electrophoresis and functional activity assays. Degradation was also not observed following exposure to hair or skin at 37 degrees C. However, incubation in fetal bovine or human sera at 37 degrees C led to degradation and loss of activity. Therefore, siRNA in the bloodstream is likely inactivated, thereby limiting systemic exposure. Interestingly, partial degradation (observed by gel electrophoresis) did not always correlate with loss of activity, suggesting that partially degraded siRNAs retain full functional activity. To demonstrate the functional activity of unmodified siRNA, EGFP-specific inhibitors were injected into footpads and shown to inhibit preexisting EGFP expression in a transgenic reporter mouse model. Taken together, these data indicate that unmodified siRNAs are viable therapeutic candidates.

Hepatitis C virus (HCV) is a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. Currently available treatment options are of limited efficacy, and there is an urgent need for development of alternative therapies. RNA interference (RNAi) is a natural mechanism by which small interfering RNA (siRNA) or short hairpin RNA (shRNA) can mediate degradation of a target RNA molecule in a sequence-specific manner. In this study, we screened in vitro-transcribed 25-bp shRNAs targeting the internal ribosome entry site (IRES) of HCV for the ability to inhibit IRES-driven gene expression in cultured cells. We identified a 44-nt region at the 3! -end of the IRES within which all shRNAs efficiently inhibited expression of an IRES-linked reporter gene. Subsequent scans within this region with 19-bp shRNAs identified even more potent molecules, providing effective inhibition at concentrations of 0.1 nM. Experiments varying features of the shRNA design showed that, for 25-bp shRNAs, neither the size of the loop (4‐10 nt) nor the sequence or pairing status of the ends affects activity, whereas in the case of 19-bp shRNAs, larger loops and the presence of a 3! -UU overhang increase efficacy. A comparison of shRNAs and siRNAs targeting the same sequence revealed that shRNAs were of comparable or greater potency than the corresponding siRNAs. AntiHCV activity was confirmed with HCV subgenomic replicons in a human hepatocyte line. The results indicate that shRNAs, which can be prepared by either transcription or chemical synthesis, may be effective agents for the control of HCV.