Mang Yu

Intracellular application of hairpin ribozyme genes against hepatitis B virus.

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.

In vitro and in vivo characterization of a second functional hairpin ribozyme against HIV-1

We have constructed a hairpin ribozyme targeted to cleave a conserved sequence in the HIV-1 pol gene. The ribozyme was modified to include a structure-stabilizing tetraloop. In vitro studies revealed a cleavage efficiency unprecedented for hairpin ribozymes (Kcat/Km = 75 min-1 microM-1). Stable retroviral vector transduction of this ribozyme gene in T-cell lines resulted in long-term ribozyme expression. As compared to control vector transduced T-cells, the pol ribozyme-transduced cells exhibited significant inhibition of different strains of HIV-1 virus production; this protection was greater when ribozyme expression was driven from an internal pol III promoter (adenovirus VA1) than when driven by a pol II promoter (the MMLV LTR). These results further demonstrate the potential of hairpin ribozymes as anti-HIV gene therapy agents and suggest possibilities for employing combinations of independently targeted hairpin ribozymes.