The Establishment Of Retrovirus-based RNA Interference Delivery System And Application In Hepatitis B Virus Gene Expression

Double-stranded RNA (dsRNA) induces sequence-specifiC posttranscriptionalgene silencing in many organisms by a process known as RNA interference (RNAi).Short interfering RNA (siRNA) is the key of RNAi. Long dsRNA can be processedto siRNA of 21-23 nucleotides (nt) by Dicer, a RNAШ-type endonuclease. Uponbinding to RNA-induced silencing complex (RISC), double-stranded siRNA isunwound and targeted to homologous mRNAs, resulting in sequence-specificcleavage and degration of mRNA.Currently, although there are several methods for generating siRNAs for genesilencing studies, they rely on transfection for delivery and cannot generatelong-term gene silencing in vivo. It is necessary to develop the more efficienttransfection system. The retrovirus is the most commonly used for the constructionof viral vectors because of its high transduction efficiency, stable insertion of a geneinto host genome, and lack of adverse host immune response to retroviral vectors.Hepatitis 13 virus (HBV) infection can cause acute or chronic viral hepatitisand other liver diseases including cirrhosis and hepatocellular carcinoma. Despitethe availability of a vaccine, hepatitis B remains a major health threat in manycountries. The use of interferon or nucleoside analogs can partially inhibit the replication of HBV. At present, there is no effective measures to dear out the virus.Therefore, the evaluation and investigation of novel strategies to treat this diseaseare necessary and urgent.In this study, retrovirus vector combined with RNA interference, a new deliverysystem of siRNA into cells was established firstly. We constructed several retrovirussystems for delivery of sip53 into human HepG2 cells. By using the single cellclones integrated with retroviral genomes in HepG2 cells, it was found that theexpression of human p53 gene in HepG2 cells couldbe substantially down-regulated.In addition, the transduction system showed that retroviral vector-mediated RNAicould allow stable inactivation of human p53 gene in established HepG2 cell clonesfor two months. The results also displayed that the retroviral vector pXSN was thesystem more effective and stable than pXRN. The second transduction withpBabe-sip53 demonstrated that the inhibition efficiency was dosage-dependent ofshort hairpin RNAs (shRNA). To our knowledge, this is the first time to identify therelationship between RNAi efficiency and the copy number of shRNA integratedinto the genome. No initial interferon response was observed when the 19complementary base pair shRNA insered into the expression vector was used. Allthese results indicated that retrovirus vector-delivered RNAi could triggerdown-regulation of p53 gene in an efficient and sequence-specific manner in HepG2cells and be used in functional genomics and in gene therapy.RNA interference (RNAi) of virus-specific genes has emerged as a potentialantiviral strategy. Herein, we developed a similar retrovirus-based RNAi systemwhich utilized the U6-RNA polymeraseⅢ(PolⅢ) promoter to drive efficientexpression and deliver the HBV-specific short hairpin RNAs (siHBVs) in bothHepG2 and HepG2.2.15(2215) cells. In this system, the retrovirus vector with aG418 or puromycin selection marker could be integrated into the host cell genomesand allowed stable expression of siHBV.In G418-resistant HepG2 cells transfected with foreign Topo-HBV plasmid, thelevels of both HBsAg and HBeAg was dramatically reduced by over 93％by usingELISA assay and the mRNA of HBV was significantly reduced based on Northern blot analysis, indicating that this RNAi system could inhibit expression ofexogenous HBV gene.In Puro-resistant 2215 cells which were stably transformed with several copiesof the HBV genome, the levels of both HBsAg and HBeAg were dramaticallyreduced by over 88% and this inhibition could last for two months as assessed byELISA. Moreover, the mRNA of HBV was significantly reduced by above 90%based on Northern blot analysis and HBV replication was markedly restrained byover 3.3-fold, as assessed by the real-time PCR. In conclusion, expression ofHBV-specific shRNAs by this RNAi system is capable of efficient and specificinhibition of expression and replication of endogenic ongoing HBV gene for a longtime.To further understand the in vivo behavior of small RNA oligos, we conducted aseries of studies to evaluate the safety, cytotoxicity, and tissue distribution ofantisense RNA oligos in cells and animals. The results showed that no obviouscytotoxicity could be found. The injection results of fluorescent-labeled oligos(FL-oligos) displayed the highest concentration of small RNA oligos in spleen, liverand kidney. On the whole, no significant toxicity associated with small RNA oligoswas observed and small RNA oligos could be widely distributed in the body.In conclusion, a stable and high specific delivery system for siRNAs mediatedby retrovirus vector was established in this study. HBV gene was efficiently andstably inhibited in vitro study based on this system specifical targeting HBV. Allabove results indicated that retrovirus-based RNAi may have a potential therapeuticapproach for chronic HBV and other persistent infections such as HCV and HIV andprovide new clues for prophylactic vaccine development.