Inhibition of hepatitis B virus replication by targeted pretreatment of complexed antisense DNA in vitro

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Abstract

We have shown that antisense oligonucleotides can be targeted to hepatitis B virus (HBV)-infected cells, resulting in specific inhibition of viral protein synthesis and replication in vitro. The targeting system was based on the internalization of DNA complexes by highly selective receptors for galactose-terminal glycoproteins, asialoglycoproteins, on the surface of hepatocytes. Our objective in this study was to determine whether antisense DNA could be targeted to hepatocytes to prevent subsequent infection by HBV. A 21-mer phosphorothioate-linked oligo DNA complementary to the HBV polyadenylation signal and 5′-upstream sequences was complexed to a targetable DNA carrier consisting of asialoglycoprotein coupled to polylysine. Pretreatment of Huh7, asialoglycoprotein receptor (+) cells, with antisense complexes before lipofection with an HBV-plasmid at a level of 6.5 × 106 copies of plasmid per cell inhibited the amount of newly synthesized, core-associated viral DNA in Huh7 cells to undetectable levels, less than 0.1 pg, as assessed by quantitative polymerase chain reaction (PCR). Hepatitis B viral RNA transcripts were decreased by 60% compared with controls as detected by RNase protection assays, and HBV surface antigen (HBsAg) accumulation was inhibited by 97%. The inhibition lasted for 6 days and was dose dependent. Controls consisting of antisense alone and a random oligo complex showed no significant effect on any of the parameters under identical conditions. We conclude that preexposure of cells to targeted complexed antisense DNA can substantially block viral gene expression and viral replication after transfection of HBV DNA.

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