Inhibition Of Hepatitis B Virus Gene Expression And Replication By Vector-based Small Interfering RNAs In HepG2 2.2.15 Cells And Transgenic Mice

Hepatitis B virus (HBV) infection is still a major global health problem, there are an estimated 350 million chronic HBV infected people worldwide, and over one million people die of chronic hepatitis, cirrhosis, or HBV-associated hepatocellular carcinoma (HCC) annually. In our coutry about 10% of population are HBV cariers. Current options to treat chronic HBV infection are restricted to the use of interferon (IFN) alfa and nucleoside analogues such as lamivudine or adefovir, but these treatments have some drawbacks, including the success of each regimen is limited because the response rates are below 30% for IFN, whereas antiviral drug-resistant strains can be selected after a long period of lamivudine therapy that may result in disease progression. It is therefore important to develop a new strategy to treat HBV patients.RNA interference (RNAi) is the phenomenon of sequence-specific, post-transcriptional gene silencing (PTGS) triggered by double-stranded RNA (dsRNA). RNAi exists in extensive organisms such as Caenorhabditis elegans, Drosophila, plants, mammalian, and is regarded as the genome's immune system and genetic watchdog among eukaryotes. Although initially recognized as a handytool to reduce gene expression, RNAi is now recognized as a mechanism for cellular protection and cleansing: it defends the genome against molecular parasites such as viruses and transposons, while removing abundant but aberrant nonfunctional messenger RNAs, as well as regulate developmental programs in a sequence specific manner. Now RNAi has developed into a novel gene-blocking tool and used to knochdown specific gene expression efficiently in various species. Its application includes screening new genes, functional genomics and gene therapy in which great achievement has been made. As far as HBV antiviral therapy is concerned, a series of reseaches had been carrid out, researchers from different laboratories successfully inhibited the HBV gene expression and replication in vitro and in vivo by introducing synthetic siRNA or vector-based siRNA targeting HBV coding sequence. In HBV established cells or animals, several variables could affect the utility of RNAi for the treatment of an ongoing HBV infection. First, in the context of an established infection, viral RNAs may be protected within complexes or nucleocapsid structures. A second variable is how efficiently preexisting viral RNA can be reduced and to what extent RNA suppression can block viral DNA replication. Finally, established viral gene expression may allow for the induction of viral RNAi-defense mechanisms. However, persistent suppression of HBV allowing the analysis of loss-of-function phenotypes that develop over longer periods of time and the effect of this suppression on host cells have never been investigated before, in addition, the therapeutic potential of nonviral vector-based siRNA for the treatment of chronic HBV infection where ongoing viral gene expression and replication are established in the liver before siRNA delivery has not been previously addressed.In our experiments, inhibition of HBV gene expression and replication in HepG2 2.2.15 cells and transgenic mice were carried out by the pSUPER RNAi system. The latter is a mammalian expression vector that directs intracellular synthesis of siRNA-like transcripts and has been used to cause efficient and specific down-regulation of gene expression, resulting in functional inactivation of the targeted genes. Based on the HBV sequence in HepG2 2.2.15 cells, five sequences targeting HBV gene were designed, synthesized 64nt oligonucleotidescontaining a unique 19-nt sequence derived from the target transcript were cloned into the RNA polymerase III based pSUPER between the Bgl WHind III sites of the vector by annealing and ligation. The constructed siRNA expression vectors were digested with EcoR I and Hind HI, and insert sequences of positive clones were confirmed by DNA sequencing. The restriction endonuclease digestion results showed that insert fragment of positive clones were consistent with what expected, DNA sequencing results identified that inserts were correct. These results demonstrated that the pSUPER vectors targeting HBV were successfully constructed. The five siRNA expression vectors were designated as pSUPER-HBVl, pSUPER-HBV2, pSUPER-HBV3, pSUPER-HBV4 and pSUPER-HBV5.The five recombinant plasmids were cotransfected with pTK-Hyg into stable HBV-producing HepG2 2.2.15 cells and single clonal cell strains were screened in 200ug/ml hygromycin media after 4 weeks. The growth curve of screened cells were made and cell cycle was analyzed by flow cytometer (FCM). HBV mRNA were detected by RT-PCR and HBV transcripts were measured by Northernblotting. Then, 3 X105 sreened cells were seeded into 6-well plates and had been maintained in 10% fetal bovine serum Dulbecco's modified Eagle medium containing 200ug/ml hygromycin and 200ug/ml G418 at 37°C under 5% CO2 for48 or 72 hours. Cell layers and media were collected, the HBsAg and HBeAg in the supernatant of the screened cell strains were assayed with Abbott MEIA Kits, the expression of intracellular viral antigens were determined by immunofluorescence staining and Western blotting. HBV DNA were qualified by real-time fluorescence quantitative PCR (FQ-PCR).The results showed that: ?A series of single clonal cell strains stable expressingsiRNA were obtained in hygromycin meadia. Flow cytometry (FCM) analysis and cellular growth curve of creened cells showed that cell cycle, proliferation index(PI) and growth velocity had no distinct difference compared with control. ?AtmRNA level, RT-PCR results showed that the HBV mRNA were markedly decreased. Northern blotting results revealed that HBV 3.5kb, 2.4kb, 2.1kb transcripts were reduced noticeably and the HBV transcripts in pSUPER-HBV2 and pSUPER-HBV5 treated cells were nearly invisible. Whereas the irrelevant pSUPER-EGFP and control did not show any inhibitory effect on HBV replicationand expression. (3)At protein level, five vector-based siRNAs could inhibit thesecretion of HBsAg and HBeAg significantly (PO.05). The suppression effect of pSUPER-HBV2 is the most potent among five siRNA expression vectors, and pSUPER-HBV2 resulted in 98% inhibition of secreted HBsAg to almost undetectable levels and 88% inhibition of HBeAg in the 72h cultured cell supernant, pSUPER-HBV5 caused 90% reduction of HBsAg and 80.8% reduction of HBeAg in the 72h supernant These results are better than publised dada in the same cell model. Immunofluorescence staining and Western blotting resultsrevealed that intracellular viral antigens were significantly reduced. ?Detection ofHBV DNA was carried out on samples from genomic DNA and culture media of screened cells by FQ-PCR. The results showed that vector-based siRNA could efficiently suppress the replication of intracellular and extracellular HBV DNA. Significant reductions of 99.7% and 98.5% extracellular HBV DNA level, 82.8% and 82.1% intracellular HBV DNA level were observed when HepG2 2.2.15 cellswere transfected with pSUPER-HBV2 and pSUPER-HBV5 respectively. (5)Dynamic analysis of HBV antigens in screened cells revealed that vector-based siRNA could sustainedly and effectively knockdown HBV gene expression: HBsAg level in culture media had remained relatively constant for 10 months, except HBeAg level slightly increased with the prolonged culture. These results demonstrate that vector-based siRNA targeting HBV can stably inhibit HBV gene expression and replication with great potency and specificity for a long time in vitro.