Inhibition Of Hepatitis Bvirus Expression And Replication By Asgpr-directed Delivery Of Sirna

【OBJECTIVE】To construct the vectors of pGenesil-siHBV1～6 expressing smallinterfering RNAs (siRNAs) targeting HBV gene sequences specifically andevaluate inhibitory effect of these siRNAs on HBV expression and replication inHepG2.2.15 cells. Then chose the best one out of these siRNAs. The higheffective vector was transfected into HepG2.2.15 cells by ASGPR-directed celltype-specific delivery and the target and inhibitory effect were detected.【METHODS】1. With the designed restriction site,siRNAs targeting the HBV gene weresynthesized and cloned into a RNA polymeraseⅢbased expression vectorpGenesil-1. The plasmid pGenesil-siHBV-HK which contains a irrelevant siRNAsequence was constructed as a control. The recombinant plasmids weretransformed into E.coli strain DH5a and positive cell clones were identified andcalled pGenesil-siHBV1～6 respectively.2. Then the recombinant plasmids were transfected into HepG2.2.15 cells byLipofectiamineTM 2000 reagent. The HBV mRNA were detected by Real-timePCR at 2d,3d and 4d post-transfection .The levels of HBsAg and HBeAg in thesupernatant of HepG2.2.15 cells were assayed with ELISA test kits at 2d,3d,5d and 7d post-transfection. Real-time PCR was used to examine the level ofcccDNA in the supernatant of HepG2.2.15 cells at 2d,3d and 4d posttransfection.3. The recombinant plasmid pGenesil-siHBV1 was transfected into HepG2.2.15 cells by jetPEI-Hepatocyte reagent. EGFP was observed by inverted fluorescencemicroscope. Then FCM was used to detect the transfection efficiency. The levelsof HBsAg and HBeAg in the supernatant of HepG2.2.15 cells were assayed withELISA test kits at 2d,3d,5d and 7d post-transfection. The intracellular HBcAgwas detected by flow cytometry and the intracellular HBsAg was assayed byimmunocytochemistry at 3 d post-transfection.The apoptosis was detected by flowcytometry at 3d post-transfection.【RESULTESULTS】1. The pGenesil-siHBV1～6 expression vectors were successfully constructed.2. All siRNAs targeted to the hepatitis B virus resulted in lower mRNA andprotein expression in HepG2.2.15 cells and the cells treated with pGenesilsiHBV1yielded the greatest inhibitory effect.3. The recombinant plasmid pGenesil-siHBV1 was transfected into HepG2.2.15cells by jetPEI-Hepatocyte reagent. EGFP were visible with inverted fluorescencemicroscope and 45% EGFP positive cells were detected by FCM. Whereas jetPEIHepatocytenegative control group had no EGFP. The levels of HBsAg,HBeAgand the intracellular HBcAg were detected to be depressed apparently,lower thanthe LipofectiamineTM 2000 reagent transfected group. The HepG2.2.15 cells haveno apparent apoptosis at 3d post-transfection.【CONCLUSION】These results demonstrate that using ASGPR-directed cell-type-specificdelivery of siRNA targeting HBV gene can inhibit HBV expression andreplication better.