title: Sequencing of PCR amplified HBV DNA pre-c and c regions in the 2.2.15 cells and antiviral action by targeted antisense oligonucleotide directed against sequence.
abstract: AIM:To study the specific inhibition of HBV gene expression by liver-targeting antisense oligonucleotide (ASON) directed against pre-c and cregious in a sequence specific manner.METHODS:According to the result of direct sequencing of PCR amplified products, a 16 mer phosphorothioate analogue of the antisense oligonucleotide (PS-ASOn) directed against the HBV U-5-like region was synthesized and then linked with one live-targeting ligand, the galactosylated poly-L-lysine.Their effect on the expression of HBV gene was observed using the 2.2.15 cells.RESULTS:HBV DNA in the 2.2.15 cells was from HBV with surface antigen subtype ayw 1 by sequencing so that antisense oligonucleotides could bind specifically to the target sequence through base piring. Under the same experimental conditions, the inhibitory rates of PS-ASON to HBsAg and HBeAg were 70% and 58% at a concentration of 10&mgr;mol/L, while by ligand-PS-ASON they were 96% and 82%, the amount of HBV DNA in cultured supernatant and cells was reduced significantly. An unrelated sequence oligonucleotide showed no effectiveness. All the oligonucleotides had no cytotoxicity.CONCLUSION:Antisense oligonucleotides complexed by the liver-targeting ligand can be targeted to cells via asialoglycoprotein receptors, resulting in supecific inhibition of HBV gene expression and replication.
authors.forename: Sen
authors.initials: S
authors.lastname: Zhoug
authors.forename: Shou-Ming
authors.initials: SM
authors.lastname: Wen
authors.forename: Ding-Feng
authors.initials: DF
authors.lastname: Zhang
authors.forename: Quan-Li
authors.initials: QL
authors.lastname: Wang
authors.forename: Seng-Qi
authors.initials: SQ
authors.lastname: Wang
authors.forename: Hong
authors.initials: H
authors.lastname: Ren