There is growing interest in the application of antisense RNA for inhibition of gene expression and potential gene therapy of viral infections. We have studied the inhibitory effect of antisense RNA directed against hepatitis B virus (HBV) genome on the expression of HBV surface antigen (HBsAg) Initial experiments were performed in vitro. Three prokaryotic antisense RNA expression constructs were made, which expressed antisense RNA complementary to the entire coding region (1.4kb), to 1.0kb of 5$\sp\prime$-region and to 582b of 5$\sp\prime$-region of HBsAg mRNA. In an in vitro translation system, all three antisense RNAs showed inhibitory effect on translation of HBsAg mRNA. Inhibition was dose-dependent with the most significant effect seen when the molar ratio of antisense RNA to mRNA was 10 to 1. In a coupled in vitro transcription and translation system, concentration-dependent inhibition of HBsAg synthesis was observed for all above mentioned antisense RNAs Next, three mammalian antisense RNA expression vectors were constructed, which expressed the same antisense RNAs as used for the in vitro system. Transfection of the vectors into Hep3B cell line, an HBsAg secreting human hepatocellular carcinoma cell line, resulted in nearly complete block of HBsAg production, while control cells (transfected with parent vector) secreted high levels of HBsAg. The inhibitory effect was long lasting, and remained demonstrable up to the time of writing of this paper (10 months posttransfection). These results suggest that antisense RNA may be used to inhibit HBsAg production by HBV infected cells To examine the probable mechanism of antisense RNA action in Hep3B cell line, we detected HBV mRNA levels in the transfected cells and found that the mRNA levels in the antisense RNA expression cells were much lower than those in the control cell. Therefore, in Hep3B cell, the antisense RNAs exerted their effect on HBsAg synthesis, at least partially, through the reduction of HBV mRNA levels 3$\sp\prime$-Truncated HBsAg has been shown to be capable of transactivating some cellular oncogenes, and therefore, may play a major role in HBV associated hepatocarcinogenesis. We hypothesized that the transformation of primary hepatocytes to the Hep3B cell line might be caused by its production of HBsAg. To examine this hypothesis, we monitored the growth rate of the cells in reduced serum conditions and observed that growth rates of cells expressing antisense RNA were significantly decreased compared to control cells in 0.5% serum containing media. We then conducted colony forming efficiency tests for the cells, and the results showed that antisense RNA expression cells formed significantly fewer colonies than control cells. The above data suggested the active role of HBsAg in Hep3B cellular proliferation
