The Screening Of Anti-HBV SiRNA And Antivirus Research In Vitro And Vivo

Hepatitis B infection is one of the most important popular heathy problem.Although the application of HB vaccine greatly reduced the incidence of Hepatitis B,more than 400 million people worldwide are persistently infected with the hepatitis B virus(HBV).The current drug treatments is still insufficient to cope with the challenges.RNA interference is a sequence-specific gene silencing mechanism in vivo found in recent years.RNAi technology has being focused on being applied including viral hepatitis and other infectious diseases therapy.And access to both high efficient and suppression of the conservative nature of the target sequences is the important foundation of the RNAi therapy.In the study,through the establishment of a multi-genotype of HBV cell model and small animal model and assistanted by bioinformatics,confirmed the siRNA target on the HBV genome with large-scale screening and verification.The study provided an important basis for further application of RNAi technology to new hepatitis B therapy.In this study,623 reported HBV genomes downloading from Genebank was done homology analysis by GCG,resulting a 95%conservative sequence.This conservative sequence was selected as the template which was employed to design siRNA sequences.Assistanted by the online tools(siRNA Off-target Search),we got 40 siRNA sequences,which were constructed to the corresponding shRNA expression plasmids on pSUPER vector,aiming to HBV genome.We detected the inhibition efficient of the 40 siRNAs using co-transfection shRNA plasmids and 5 HBV 1.3 genome that belong to genotype C1,D1 and Ae and can express HBsAg and HBeAg effectively in Huh7 cell.Through the analysis in transfection,we established the best time point for detection and the community plasmid transfer ratio.Then we cotransfected shRNA expressing plasmids and HBV 1.3 genome at the 1:10 ratio to Huh7 cell and evaluated the inhibition ability of the 40 designed siRNA.The result indicated all of them can inhibit the HBV in varying degrees,some of them are very effective in inhibiting HBV(Some targets have applied for patents,the application No:200810110686.4).We chosed 3(B244,B245 and B376) for further research and verification.The result in CCK8 test and IFN related mRNA test suggested that all of the three siRNA have no cytotoxicity and can not induce off-target effects.Another important problem of RNAi therapy for anti-virus focuses on delivering RNAi element into the target cells effectively.We studied the feasibility of lentiviral vector and chemical modification on siRNA delivery.We constructed some shRNA on lentiviral vector and confirmed the recombinant lentivirus with shRNA element inhibiting HBV effectively through infecting the HepG2-N10 cell.On the other hand, siRNAs with chemical modifications include phosphorothiolation of the non-bridging oxygen and cholesterol modification at the 3'end,2'sugar modification has better effect on HBV inhibition.A mouse model of acute HBV infection was developed successfully using hydrodynamics to validate the effect of anti-HBV siRNA in vivo.Through co-injfection 5 HBV 1.3 genomes and shRNA expression plasmids(B244,B245 and B376),the significantly reducing of HBsAg,HBeAg and HBV DNA expression indicated that the siRNA we got inhibited the replication and expression of various HBV of different genotype.