Esirna Inhibition Of Hbv Replication

RNA interference, the process of specific mRNA degradation induced by homologous dsRNA, has been widely used for target-specific gene silencing, and holds great therapeutic potential in clinical applications, especially in the field of anti-cancer and anti-virus therapy. We have previously reported a cost-effective method to prepare esiRNAs in Escherichia coli based on cloning, fermentation, digestion and purification. We have proved that esiRNAs prepared with this method can specifically and effectively inhibit the replication of hepatitis B virus in human liver cancer cells.In this thesis, we first described a modified dsRNA expression system which can improve the yield of dsRNA. Four different types of esiRNAs targeting the four open reading frames (ORFs) of human Hepatitis B virus (HBV) were prepared by using this new method. Their capacity to inhibit HBV replication was tested in HepG2 cells, and the esiHBVP targeting the HBV DNA polymerase gene was the most potent one. Further more, esiHBVP can inhibit HBV replication in mice more efficiently than chemically synthesized siRNA. In order to test whether the esiHBVP can inhibit Hepatitis B virus of different genotypes, we amplified the esiHBVP target sequence from the serum of HBV positive patients, and cloned them to the 3' untranslated region (3' UTR) of a report system. In vitro experiment showed that the esiHBVP can inhibit the report gene expression with almost same efficiency regardless of the similarity of the target sequences. We then prepared esiHP9 based on the esiHBVP target sequence derived from HBV genotype B, which shares 87% similarity to HBV genotype C gene sequence. In vitro and in vivo experiment showed that the esiHP9 also can inhibit HBV genotype C as well as esiHBVP that is 100% similar to the sequence of HBV genotype C. The above results suggest that esiRNA can tolerate target sequence variation to some extent without losing its inhibitory capacity. The results also imply that esiHBVP might be able to inhibit the replication of both HBV genotype B and C with high efficiency, which are the main genotypes of HBV that infect Chinese population.In the first chapter of this thesis, we developed a modified dsRNA expression system and also improved the esiRNA purification procedure. We also proved that the esiRNAs prepared by the new procedure could specifically inhibit homologous gene expression without activating the IFN system. In the new expression system, the senseand anti-sense strand of the target gene were cloned into an expression vector separated by a gap sequence. The gap sequence is indispensable to make the expression vector stable. After transcription, the sense and anti-sense strand of the target gene match together to form stable dsRNA immediately, which greatly improved the yield of dsRNA for about 5 fold compared with the two opposing promoter expression vector.In the second chapter, we proved esiRNA is more efficient than chemically synthesized siRNA to inhibit the replication of HBV in vivo. Four different types of esiRNAs targeting the four ORFs of HBV gene were prepared. Their capacity to inhibit the replication of HBV was tested in HepG2 cells, and the esiHBVP was the most potent one. The capacity of the esiHBVP to inhibit the replication of HBV in mice model was compared to that of the most potent siRNA reported in literature. It turned out that the esiHBVP was more potent than the synthesized siRNA. A single dose of 1 μg of esiHBVP was able to reduce the expression level of HBsAg and HBeAg in the mouse serum by 90% and 89% one day after injection, while the same amount of chemically synthesized siRNA only reduced that by 33% and 45%. Quantification of HBV DNA in the mouse serum showed that the treatment of 1 μg eiHBVP could reduce serum HBV DNA copy number to 18% that of the untreated control, while 1 μg siRNA treatment only reduced that to 63%.In the third chapter, we tried to answer if the esiHBVP can inhibit Hepatitis B virus of different genotypes. We amplified the esiHBVP target sequences from the serum of HBV positive patients, and cloned them to the 3' untranslated region (3' UTR) of a report system. In vitro experiment showed that the esiHBVP can inhibit the expression of report gene with almost the same efficiency regardless of the similarity of the target sequence. We then prepared esiHP9 based on the esiHBVP target sequence derived from HBV genotype B, which shares 87% similarity to genotype C gene sequence. In vitro and in vivo experiment showed that the esiHP9 also can inhibit HBV genotype C as well as esiHBVP that is 100% similar to the sequence of HBV genotype C. The above results suggest that the esiRNA can tolerate target sequence variation to some extent without losing its inhibitory capacity. The results also imply that the esiHBVP might be able to inhibit the replication of both HBV genotype B and C with high efficiency, which are the main genotypes of HBV that infect Chinese population.