Hammerhead ribozyme-mediated inhibition of hepatitis B virus X gene expression in cultured cells

Background/Aims: Chronic infection with hepatitis B virus (HBV) is endemic to sub-Saharan Africa and parts of Asia. Common complications of HBV persistence include cirrhosis and hepatocellular carcinoma (HCC). Present treatment of chronic HBV infection is usually ineffective and novel therapeutic approaches arean important objective. The HBV X protein (HBx) is a transcriptional activator that is required for the establishment of HBV infection and is implicated in hepatocarcinogenesis. The aim of this study was to assess the ability of two endogenously expressed hammerhead ribozymes to inhibit expression of HBV genes in transfected cultured cells. Methods: Eukaryotic expression plasmids producing two ribozymes targeted to the HBx open reading frame, as well as their catalytically inactive homologues, were generated. Established cell lines and a primary culture of malignant hepatocytes were transfected to assess ribozyme effects on HBx expression and HBV replication. Results: The ribozyme-expressing vectors inhibit expression of functional HBx protein and decrease HBV mRNA encoding surface and HBx sequences in transfected cells. Moreover, decreased HBsAg and HBeAg secretion from cells transfected with the ribozymes and an HBV replication competent plasmid provide evidence for an antireplicative effect of the ribozymes. However, the data do not exclude a dominant antisense effect that inhibits HBV gene expression. Conclusions: Inactivation of HBx, a sequence that is conserved in mammalian hepadnaviruses and found in all HBV transcripts, has potential for the treatment of chronic HBV infection.