1034. Expression of Short Hairpin RNAs by Liver and Non Liver Specific RNA Pol II Expression Cassettes: What Governs Activity?

Abstract

Top of pageAbstract Infection with the Hepatitis C virus (HCV) represents a major world-wide health problem. HCV is a liver-tropic RNA virus, and RNAi based treatments have been proposed as a new therapeutic modality. We are developing short-hairpin RNAs to elicit an RNAi response against the HCV genome. Although robust expression of shRNAs from ubiquitous Pol III promoters has been demonstrated by various groups, it would be advantageous to use liver specific Pol II promoters to help restrict expression to affected tissues. We have previously demonstrated modifications that enable the Pol II liver specific apolipoprotein E-human alpha antitrypsin enhancer/ promoter expression (ApoE/hAAT) cassette expressing shRNA to exhibit up to 70% inhibition. We have analyzed the expression profiles of Pol III and Pol II driven cassettes that express identical DNAdirected shRNAs against HCV targets, and observe that Pol III promoters express shRNAs at 10-30 fold higher levels than Pol II. In our subsequent studies, it has become apparent that the modifications to shRNA generated to attain maximal inhibition from the ApoE/hAAT promoter cassette are not universally applicable to all liver specific promoters. In order to effectuate a strategy which may be more generally applicable for the generation of active PolII driven shRNAs, we designed a strategy based on the insertion of the guide strand into a pri-microRNA cassette by retaining the identical structural features (such as loops and bubbles) and physical characteristics (such as free energy delta G) of the pri-microRNA. Analysis of the first CMV IE promoter driven constructs suggests that this is a viable approach to highly efficacious PolII driven expression of shRNAs. We are presently adjusting these cassettes to liver specific expression.