Objective: To study inhibitory effect of antisense oligodeoxynucleotide (asODN) on HCV in vitro.
Methods: The H9 cells transfected by pCD-HCV, a recombinant HCV containing total HCV structural gene, were treated with two 15-mers phosphorothioate (PS) ODNs complementary (PS-ASON) and homologous (PS-ODN) to HCV core genomic region, which were labeled with digoxin (DIG). Spot blot hybridization was carried out. Treated by the two ODNs, rPS-ODN (a 15-mers PS ODN of random sequence) or PS-ASON were modified with two liposomes (DOTAP and Lipofectin) and calcium phosphate precipitation respectively. With a half-ration, the variation of level of HCV mRNA and HCV antigen expression was observed by RT-PCR and dot ELISA. 3H-TdR adding test was done to observe PS-ASON cytotoxicity.
Results: PS-ODN and PS-ASON were detected in the H9 cells. The target gene was hybridized to PS-ASON and PS-ODN labeled with DIG. PS-ASON cut down level of HCV mRNA and HCV antigen expression obviously. However, PS-ODN and rPS-ODN did not influence the level of the both. The time-dependent and dose-dependent inhibition of PS-ASON was observed. In contrast to free PS-ASON, both of liposomal PS-ASON showed more highly effective inhibition, but calcium phosphate precipitation-PS-ASON complex did not. The results showed PS-ASON did not influence the H9 cells growth at 10 mumol/L.
Conclusion: PS-ASON complementary to HCV core gene is asODN and exerts antisense-inhibitory effect on the level of HCV translation obviously, but not on the level of HCV replication and transcription.