Assessing the efficacy of RNA polymerase II- and RNA polymerase III promoter-driven RNA interference effecter cassettes targeted to HBx

摘要

Globally, the status of chronic infection with hepatitis B virus (HBV) is increasing, with approximately twenty million new cases being reported each year. Chronically infected individuals are at risk of developing complications of cirrhosis and hepatocellular carcinomas. Currently, treatment of HBV entails a combination of therapeutics and successful treatment is limited to approximately 20% of patients. Efficient inhibition of HBV replication has been shown by harnessing the RNA interference (RNAi) pathway through the utilisation of U6 RNA Polymerase III (Pol III) promoter-driven short hairpin RNA (shRNA). The generation of a double stranded RNA (dsRNA) as a therapeutic tool is a realistic approach to treating HBV, however the expression of the dsRNA requires optimisation to prevent any toxic effects associated with off-targeting or saturation of the endogenous RNAi pathway. Two Pol III (H1 and tRNALys3) promoter-driven shRNA expression cassettes and an Pol II (CMV) promoter-driven microRNA shuttle were generated and compared to the U6 shRNA constructs. Knockdown was assessed for five different target sites at varying concentrations of effecter to target ratios. The tRNALys3 shRNA 5 caused significant inhibition of HBV, whilst the H1 and cytomegalovirus (CMV) promoter-driven effecter cassettes were only effective at high target to effecter ratios (1:10). In vivo analysis in a murine hydrodynamic injection model supported the data from cell culture analysis, and established U6 shRNA 5 and tRNALys3 shRNA 5 as the most effective promoter-driven cassettes with which to silence HBV replication. Harnessing of the endogenous RNAi pathway to control the expression of HBV genes by target specific mRNA degradation is becoming an increasingly valuable approach in designing a therapeutic antiviral construct, particularly with a construct as effective as tRNALys3 shRNA5.