Inhibitory Activity Against Hepatitis B Virus (HBV) By A Novel Bifunctional SiRNA

Hepatitis B virus (HBV) infection severely endangered human beings, and no effective treatment could be conducted to the infected people or carriers. In order to develop new anti-HBV drugs, we designed a new type of bifunctional siRNA (5’-ppp-siRNA,3p-siRNA) nucleic acid drugs by stimulating the expression of antiviral active IFN-Ⅰ induced by RIG-Ⅰ, and suppressing HBV viral replication silenced by RNAi to HBV gene expression.3p-siRNA forms the RNA induced silencing complex (RISC) via being combined with a ribozyme, which silences the expression of specific genes by cleaving its complementary sequences of target mRNA induced by RISC. Meanwhile,3p-siRNA activates retinoic acid inducible gene Ⅰ (RIG-Ⅰ), interacting with mitochondria-associated IPS-1(IFN-β promoter stimulator-1)/CARDIF(CARD adaptor inducing IFN-(3)/MAVS(mitochondrial antiviral signaling protein)/VISA (virus-induced signaling adaptor), and the cascade signal activates NF-κB (nuclear factor-κB) or IRF3(interferon regulatory factor-3) to induce the transcription and secretion of Type Ⅰ IFNs and inflammatory cytokines.After3p-siRNA was generated by in vitro transcription, we found that the cytotoxic effects of the3p-siRNA on the cell viability of HepG2.2.15cells have no significant difference at concentrations of100nM and200nM, but have some change at the concentration of500nM. Moreover, the HepG2.2.15cells were co-transfected with a reporter gene of IFN-β Firefly-luciferase cassette and RIG-Ⅰ (Renilla-luciferase cassette was used as an internal control). Then,3p-siRNA transfected cells after24hours transfection, and luciferase activity was detected after another16hours. We found that the3p-siRNA only weakly induced IFN expression when RIG-Ⅰ was not transfected, but when RIG-Ⅰ co-transfected the3p-siRNA significantly induced IFN expression, suggesting that3p-siRNA has antiviral activity dependent on RIG-Ⅰ in the HepG2.2.15cells. On the other hand,3p-siRNA removed5’-triphosphate groups by CIAP were co-transfected with IFN-β Firefly-luciferase cassette and RIG-I in HepG2.2.15cells and then IFN-β luciferase activity was detected. However, phosphatase-treated3p-siRNA did not activate RIG-Ⅰ-induced IFN pathway, indicating that5’-triphosphate groups have indispensable role in3p-siRNA induced IFN expression. Additionally,3p-siRNA has RIG-Ⅰ dependent activating effects to the interferon stimulated regulatory element (ISRE) as well. The3p-siRNA weakly induced ISRE without RIG-Ⅰ co-transfected, but3p-siRNA significantly induced ISRE with RIG-I co-transfected.Antiviral activity assay showed that3p-siRNA exhibited a more pronounced antiviral activity in HepG2.2.15cells compared with siRNA.3p-siRNA had obvious suppression effect to HBsAg, HBeAg, HBV DNA, and HBV mRNA after48h transfection. Furthermore,3p-siRNA had sustained antiviral effects:after3p-siRNA transfection for48h,96h, and144h, rates of the inhibition to HBsAg and HBeAg were55%and41.8%,37%and24.3%,33.3%and14.8%, respectively; while the inhibition rates were66.3%and53%,55.5%and49.7%,45.6%and26.5%, respectively, when RIG-Ⅰ and3p-siRNA were co-transfected. Compared with mere3p-siRNA transfection, co-transfection of3p-siRNA and RIG-Ⅰ showed that3p-siRNA exhibited a more obvious inhibition activity to HBV. Additionally, the suppression of3p-siRNA to HBV presented dose dependency when3p-siRNA transfected HepG2.2.15cells with different doses (50nM,100nM,200nM).In summary, we designed a new bi-function anti-HBV nucleic acid drugs3p-siRNA.3p-siRNA silenced specific genes by specific recognition and targeted cleavage of target HBV mRNA. On the other hand, it activates the RIG-Ⅰ-mediated antivirus immune response of host cells and thus efficiently blocked HBV replication.3p-siRNA with double functions contributes to the development of new antiviral drugs.