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The deviant poly(A) signal of hepatitis B virus (HBV) not only controls the formation of the 3 end of all the viral RNA, but is also crucial for HBV replication. Hence, a cisreleasing hammerhead ribozyme (RzA) targeted to the poly(A) signal region of HBV subtype adr was investigated for its antiviral effects. In vitro, RzA cleaved HBV RNA at its target site up to 70%, while the disabled ribozyme (dRzA), which had a onebase mutation in the catalytic site, did not cleave the target RNA at all. When the ribozymes were cotransfected into HepG2 cells with the HBV genomecontaining plasmid p3.6II, the wildtype ribozyme RzA could effectively decrease HBV RNA levels and inhibit HBV replication, whereas its disabled form, dRzA, had much weaker effects, indicating that the active catalytic domain of the hammerhead ribozyme could markedly increase the extent of antisensemediated inhibition. In addition, there was a gradient of effectiveness: the higher the amount of released ribozyme, the more the reduction in target HBV RNA in cells as well as progeny DNA reduction. These results suggest the possibility of the hammerhead ribozyme RzA to be used for the gene therapy of HBV infection.

Inhibition of Hepatitis B Virus by Hammerhead Ribozyme Targeted to the Poly(A) Signal Sequence in Cultured Cells