Immunostimulatory siRNA with a uridine bulge leads to potent inhibition of HBV and activation of innate immunity

Abstract

Background Hepatitis B virus (HBV) infection is difficult to cure. HBV-specific immune tolerance plays a key role in HBV persistence, and enhancing cellular and humoral immunity will improve the control of HBV infection. The purpose of the study was to explore the anti-HBV and immunostimulatory effects of msiRNAs that introduce unpaired uridine bulges in the passenger strand. Methods msiRNAs targeting the HBV S and X genes were designed and named msiHBs and msiHBx, respectively. HepG2 cells were cotransfected with siRNA or msiRNA and the HBV replication-competent plasmid pHY106-wta or pHY106-X15. HepG2.215 cells were transfected with siRNA or msiRNA. The levels of HBsAg, HBeAg, and the cytokines TNF-α, IFN-α, IFN-β, IL-1α, and IL-6 in the culture supernatant was detected by ELISA. The levels of intracellular HBV RNA, nuclear HBV replication intermediates, and HBV DNA in the supernatant were measured by quantitative RT-PCR and PCR. The levels of HBV replication intermediates were detected by Southern blotting. Peripheral blood mononuclear cells were transfected with siRNA or msiRNA, and the levels of secreted cytokines IFN-α and IFN-β were detected by ELISA. The bioactivity of type I interferons in the supernatants was detected by the virus protection assay. Results msiHBx treatment led to a significant decrease in HBsAg (to a negative level) and HBV DNA (95.5%) in the supernatant and intrahepatocellular HBV replication intermediates (89.8%) in HepG2 cells with transient HBV replication and in HepG2.2.15 cells. There was no significant difference between msiHBx and siHBx in terms of the reduction in HBV proteins and HBV replication (P > 0.05). Compared with siHBx, msiHBx treatment of HepG2 cells transfected with the HBV replication-competent plasmid led to a significant increase in the levels of the antiviral cytokines TNF-α (3.3-fold), IFN-α (1.4-fold), and IFN-β (2.5-fold) (P < 0.01), without upregulation of the proinflammatory cytokines IL-1α and IL-6. The virus protection assay results showed msiHBx-mediated type I interferons effectively protected L929 cells against ECMV infection. Conclusions msiHBx could effectively inhibit HBV expression and replication and induce an antiviral innate immune response without proinflammatory activation. The dual RNAi and immunostimulatory activity of msiRNAs may play an important role in the control of HBV infection.

PreS/2-21-Guided siRNA Nanoparticles Target to Inhibit Hepatitis B Virus Infection and Replication

“…The results of HE staining of the heart, liver, spleen, kidney, brain and lung sections from the PSN-treated mice showed that PSN intervention could improve liver injury in HBV-infected mice without other organ damage, which indicated that the efficacy and safety of PSN were not in doubt. For the antiviral effect of PSN, we found that the levels of HBV DNA, HBeAg and HBsAg in the PSN-treated hepatitis B mice group were significantly lower than those in the model group, which is consistent with the results of related studies that siRNA can significantly reduce the levels of HBeAg and HBsAg in vivo (7,38). Furthermore, we discovered that PSN lowered ALT and AST levels in hepatitis B mice, confirming the antiviral action of PSN in vivo and improving liver function to some extent.…”

“…The standard curve for HBV DNA was plotted using Ct values and the copy number was converted to international unit IU/mL. After centrifuging the supernatant, 50 mL was transferred to a new PCR tube, heated at 100°C for 2 min, then 1 mL was taken into the qPCR system (7), and the level of HBV DNA was calculated using the standard curve. After centrifugation to remove cell debris, the supernatant was obtained and HBsAg ELISA kits (Keygen Biotech, Co., Ltd) were used to detect HBV antigen levels according to the kit instructions.…”

“…RNA interference (RNAi) is a gene therapy that can be used to inhibit HBV replication and treat hepatitis B by mediating targeted mRNA degradation or mRNA translation inhibition to silence the expression of target genes in a specific manner (5). Clinical studies with siRNA drugs targeting the Pre-C, Pre-S1, Pre-S2, and X genes of HBV are now underway, with promising results (6,7). Since all five mRNAs transcribed by HBV contain the X gene sequence, siRNA targeting the X gene can simultaneously inhibit the in vivo replication of HBV by inhibiting the formation of pregenomic RNA (pgRNA).…”