Inhibition Of HBV Replication By Adenovirus-mediated RNAi In Vitro

There are about 3.5million people who are suffering chronic HBV (hepatits B virus, HBV) infection in the world. Chronic HBV infection, which can lead to the development of liver cirrhosis and hepatocellular carcinomar, is currently treated with interferon or nucleoside analogs such as Lamivudine, Entecavir and Adefovir dipivoxil. The success rate of these treatments, however, is low and frequently reinfectious occur.RNA interference (RNAi) is a recently discovered technology of silencing gene. It is defined as 21-23bp small interfering RNA (siRNA) inducing the sequence-specific degradation of homologous messenger RNA (mRNA) which results in gene silencing at the post-transcriptional level. It has shown that 21-23bp siRNAs induce efficient RNAi in various mammalian cells without activating the non-specific response that generally suppress mRNA translation when cells exposure to dsRNA greater than 30 bp in length. Thus RNAi-mediated gene silencing offers a potentially powerful tool to inhibit HBV replication.RNAi-mediated viral suppression is fundamentally distinct with conventional drug therapies in that it inhibits viral gene expression. RNAi can target at multiple conserved viral elements, minimizing the chances of viral escape. It has been proved RNAi can inhibit HBV replication both in vitro and vivo. RNAi-based therapies would be predicted to be an alternative treatment of HBV infection. However, an ideal vector that can transfer siRNAs effectively and safely to liver remains a problem for RNAi-based therapy used in clinical.Molecular biology has developed rapidly in recent years. People have learned more about the structure and function of viral genome and the mechanism of viralinfection. The technology of constructing viral vector has been improved. As one of the most widely used viral vector, adenoviral vector can transduce a wide spectrum of cell types and do not require division of the target cell for gene transfer or expression. The adenovirus chromosome remains episomal in the transduced cell, thus avoiding the possibility of insertional mutagenesis.In this study, we constructed a recombinant adenovirus expressing both GFP and shRNAs, which can effectively inhibit HBV replication in HepG2.2.15 cells- an ongoing viral replication model. Further studies will be continued on the basis to determine the transfer efficiency and possible side effects of the adenovirus-transferred siRNAs in animals. Our aim is to discover a possible vector that can be used in HBV clinical therapy.Inhibition of hepatitis B virus by adenovirus-mediated RNAi in vitro1, Construction and identification of the shuttle plasmid pShuttle+GFP+shDNAA plasmid without HindIII sites was acquired by digested plasmid pEGFP-C1 with endonuclease BglII and BamHI and reconnected. The GFP (green fluorescent protein) expressing cassette containing CMV promoter was obtained by PCR. The PCR product was inserted into the plasmid vector after digested. The recombinant plasmids were screened and identified by PCR and transfection into HEK293 cells. Primers were designed to amplify the gene can expressing shRNA (small hairpin RNA) targeting the 579-597sites of HBs gene by U6 promoter from plasmid pAV6. the PCR products were also cloned into the plasmid pShuttle+GFP and the recombinant plasmid pShuttle+GFP+shDNA was identified by PCR and DNA sequences analysis.2, Construction and identification of the recombinant adenovirusThe recombinant plasmid pShuttle+GFP+shDNA was linearized by restricted endonuclease PmeI and transformed into BJ5183 cells to produce recombinant adenovirus(rAd) plasmids under 200Ω , 2.5KV , 25μF. The recombinantadenovirus(rAd) plasmids were identified as positive by PacI digestion and then were transformed into DH5a cells to gain more plasmids.3,Package, amplification of the recombinant adenovirus and viral titer evaluationThe purified rAd plasmids were linearized with PacI and subsequently transfected into HEK293 cells to product rAd viral particles. Further amplification of virus primary stocks was achieved by infection in HEK293 cells. Finally, we transfected HEK293 cells with different MOI. The expression of GFP by rAd was detected by flow cytometry 30h posttransfection, thus the virus particle titre was determined.4,The influence of adenovirus-mediated RNAi with different MOISecretions of HBsAg and HBeAg in HepG2.2.15 cells infected with different MOI of recombinant adenovirus were monitored by Radioimmunoassay(RIA) at day 2,5,8 and 11 post infection. The supernatant of HepG2.2.15 cells was also collected for HBV DNA detected at day 2,5,8 and 11 post infection. At day 2, cells were collected to count the cells expressing GFP by . HBV RNA was detected at day 4 post infection.5,Detection of HBV protein by Radioimmunoassay analysis (RIA)After the recombinant adenovirus infected cells witn MOI 80 and 160 respectively, the concentrations of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells were detected by RIA. It showed the concentration of cells supernatant infected with AD-GFP-ShDNA were obviously reduced compared to the control. The inhibition reached peak at day 8 post transfection with the HBsAg inhibition rates were 64% (MOI=80), 92% (MOI=160) and HBeAg inhibition rates were39% (MOI=80), 61% (MOI=160) respectively. The inhibitory effects lasted at the observed 11 days.6,The influence of adnovirus-mediated RNAi on HBV transcripts by RT-PCRThe HBV RNA was extracted from infected cells at day 4 post transfection and the cDNA produced by reverse transcription(RT) was used as a template for PCR with primers designed in HBc gene . As a control, primers in β-actin gene were also designed. The results showed the lighteness of the objective band was obviously darkcompared to the control.7,The influence of adnovirus-mediated RNAi on HBV replication by real-time PCR HBV Dane particles were harvest 2,5,8, and 11days post transfection. The HBV DNA was extracted from these particles and was amplified by real-time PCR. The copies of HBV DNA were stably increased and reached peak at day 11 in both groups. The inhibition of HBV DNA was first observed at 5 day post transfection and ascended until day 11 post transfection.Conclusion(1),The replication-defective recombinant adenovirus Ad-GFP-shDNA expressing GFP and shRNA was successfully constructed.(2),The replication-defective recombinant adenovirus Ad-GFP-shDNA inhibited HBV replication and the expression of viral proteins in HepG2.2.15 cells.