siRNA pool targeting different sites of human hepatitis B surface antigen efficiently inhibits HBV infection

Abstract

The main objective was to determine whether a pool of siRNAs targeting different regions of hepatitis B virus surface antigen (HBsAg) efficiently inhibits HBV infection. siRNAs targeting different regions of HBsAg were transfected into HBV-producing HepG2.2.15 cells and at 72h post transfection, the culture medium was collected for ELISA to determine HBsAg, while total RNA was isolated from the cells for real-time PCR. Three siRNA sequences which efficiently inhibited HBV infection were converted into small hairpin RNAs (shRNAs) and then cloned into a single plasmid psiSTRIKE™ driven by a single U6 promoter. These shRNA expressing plasmids were tested for HBsAg gene silencing in HepG2.2.15 cells. A pool of siRNAs targeting HBsAg efficiently inhibited HBV replication and antigen expression when transfected into HepG2.2.15 cells, compared to the use of single siRNA. Similarly, the plasmid encoding three different shRNAs driven by a single U6 promoter was more effective in silencing HBsAg at DNA, mRNA and protein levels compared to the plasmid encoding single shRNA. No apoptotic change was observed in the cells when the plasmid was transfected at a dose of 0.5-2μg/1×10 6 cells after complex formation with Lipofectamine LTX™. Furthermore, transfection with siRNA or shRNA did not increase IFN-γ release, suggesting no induction of interferon response. In conclusion, a pool of chemically synthesized siRNAs as well as the shRNA expression plasmid encoding multiple shRNAs targeting different regions of HBsAg showed high gene silencing in HepG2.2.15 cells.

Bipartite vectors for co‐expression of a growth factor cDNA and short hairpin RNA against an apoptotic gene

“…In addition, most of the algorithms for shRNA sequence design actually comprise that of siRNA design and convert the siRNA into the shRNA sequence. In the present study, we have converted a potent siRNA targeting sequence into a shRNA sequence with success 7, 11. However, we also observed a loss of the silencing effect after converting the potent siRNA sequence into shRNA (data not show).…”

“…RNAi has been extensively used as a tool for gene silencing in cell lines and animal models 5, 6. In addition, it is also a promising approach for treating various acquired and genetic diseases 7, 8. Recently, we have tested the effects of siRNAs against inducible nitric oxide synthase (iNOS) genes on insulin producing β‐cell line (INS‐1E) and on human islets 9, 10.…”

Epigenetic Regulation of Hepatitis B Virus Infection

“…Following transfection of this shRNA, a 12-fold reduction of viral DNA after 30 passages over 8 months suggested reduced replicative capacity. Second, recent efforts have shown that the use of combination shRNAs is synergistic with respect to decreased production of HBsAg [40] as well as HBeAg, viral DNA, and cccDNA [41], and this strategy may prove useful to prevent against drug resistance. Third, these findings have been replicated in vitro.…”

RNA interference for improving the outcome of islet transplantation

“…In another study, Chen et al used siRNA pool targeting different regions of human hepatitis B surface antigen. The treatment of this siRNA pool showed efficient inhibition of hepatitis B surface antigens production at HBV-producing HepG2.2.15 cells [182]. Alternatively, co-silencing of multiple genes with siRNAs has also been used to achieve synergistic effect.…”