Specific inhibition of hepatitis b virus gene expression by an antisense oligonucleotide in vitro

It was previously shown that a number of antisense oligonucleotides against hepatitis B virus (HBV) mRNAs were highly effective in inhibition of HBV gene expression (Yao et al., 1995). Here, using radioisotope techniques, we report a specific inhibition of HBV surface antigen (HBsAg) production in vitro by 2.2.15 cells (Hep-G2 cells transfected with HBV genome) by the antisense oligonucleotide 15-S-asON, a 15-mer phosphorothioate analogue complementary to the cap site of the SPII promoter of HBV mRNA, at a concentration of 2-5 mumol/l. After 24 and 48 hrs of incubation of cells with 15-S-asON, the intracellular concentration of the latter rose to 69.4 and 75.8 nmol/l, respectively, and the HBsAg level assayed by ELISA was reduced by 50.0% and 70.6%, respectively. These results were checked by use of the radioimmunoprecipitation method: 2.2.15 cells exposed to 15-S-asON and labelled with [35S]-methionine for 48 hrs showed a decrease of the HBsAg level by 81.26% but almost none of the total proteins. No cytotoxicity of the 15-S-asON was observed with regard to the cell morphology and growth. These results indicate that the tested antisense oligonucleotide specifically inhibits the HBV gene expression.