Intracellular application of hairpin ribozyme genes against hepatitis B virus

Abstract

HBV, a partially double-stranded DNA virus, replicates through a pregenomic RNA (pgRNA) intermediate, which provides a therapeutic opportunity for a novel antiviral gene therapy based on ribozyme RNA cleavage. Three hairpin ribozymes (Rzs) were designed which have the potential to disrupt HBV replication by targeting the pgRNA as well as specific mRNAs encoding the HBV surface antigen (HBsAg), the polymerase and the X protein. The ability of each ribozyme to cleave approximately 0.3 kb HBV subgenomic RNA fragments was tested in vitro. Two of the three Rzs tested (BR1 and BR3) were capable of cleaving their respective RNA substrates, while their catalytically disabled mutated counterpart Rzs were not. Structural modifications were performed on these two Rzs, with the goal of increasing catalytic efficiency both in vitro and in cells. To determine the Rz activities in liver cells, the cDNAs for each of the anti-HBV Rzs (and their catalytically disabled negative controls) were cloned into retroviral vectors. Unmodified ribozymes co-expressed with HBV in human liver Huh7 cells reduced the level of viral particle production by up to 66% based on the endogenous polymerase assay, while the structurally modified ribozymes inhibited HBV production up to 83%. These encouraging results indicate the feasibility of ribozyme-mediated gene therapy for the treatment of HBV infections.