Combined antiviral activity of interferon-alpha and RNA interference directed against hepatitis C without affecting vector delivery and gene silencing

Abstract

The current standard interferon-alpha (IFN-alpha)-based therapy for chronic hepatitis C virus (HCV) infection is only effective in approximately half of the patients, prompting the need for alternative treatments. RNA interference (RNAi) represents novel approach to combat HCV by sequence-specific targeting of viral or host factors involved in infection. Monotherapy of RNAi, however, may lead to therapeutic resistance by mutational escape of the virus. Here, we proposed that combining lentiviral vector-mediated RNAi and IFN-alpha could be more effective and avoid therapeutic resistance. In this study, we found that IFN-alpha treatment did not interfere with RNAi-mediated gene silencing. RNAi and IFN-alpha act independently on HCV replication showing combined antiviral activity when used simultaneously or sequentially. Transduction of mouse hepatocytes in vivo and in vitro was not effected by IFN-alpha treatment. In conclusion, RNAi and IFN-alpha can be effectively combined without cross-interference and may represent a promising combinational strategy for the treatment of hepatitis C.

Fig. 1

Inhibition of HCV replication by IFN-α or LV-shNS5B. a Huh7-ET replicon was used for testing HCV replication by monitoring luciferase activity. IFN-α treatment inhibits viral replication in a dose-dependent manner. Profound reduction of luciferase activity (97 ± 2% inhibition, mean ± SD, n = 9) was observed from 2.5 to 100 IU/ml concentration of IFN-α. b LV-shNS5b contains both GFP reporter gene and shRNA targeting HCV were tested. Huh7-ET treated with increasing dose of LV-shNS5b resulted in higher levels of transduction efficiency and inhibition of HCV replication, monitored by GFP-positive population and luciferase activity, respectively. Maximum inhibition of HCV replication was observed at high dose (20 or 25 MOI) by 98 ± 3% (mean ± SD, n = 6). *P < 0.01 (Wilcoxon test) significantly different from untreated conditions

Fig. 2

Effect of exogenous IFN-α on lentiviral vector transductions in vitro and in mice. a LV-GFP vector expressing GFP under control of CAG promoter was used for transduction of Huh7 cells. b Representative histogram of GFP expression determined by flow cytometry of control cells and transduced cells 3 days after culture. c No significant (P > 0.05) differences of transduction efficiency, compared with nontreated control group, were observed with 1, 10, or 100 IU/ml of IFN-α for high (9 MOI), intermediate (3 MOI), and low (1 MOI) vector concentrations. Shown is the mean ± SD of four independent experiments. MOI multiplicity of infection. d Mice treated with exogenous IFN-α 6 h before and 3 days after administration of LV-GFP (suboptimal dose, 5 × 10⁶ transducing units) showed comparable transduction efficiency in the liver (1.7% to 4.1% transduced hepatocytes), compared with the group injected with vector only (2.1% and 4.7% transduced hepatocytes). Percentage of GFP-positive cells was indicated in the FACS picture for each individual mouse

Fig. 3

IFN-α does not interfere with RNAi-mediated knockdown of GFP. a LV-shGFP vector containing a shGFP cassette driven by U6 promoter were used. b Control (Huh7) and stable GFP expressing cell line (Huh7-GFP) were treated with LV-shGFP. GFP expression was measured by flow cytometry 3 days post- transductions, clearly showing inhibition of GFP expression by LV-shGFP in Huh7-GFP cells. c The relative GFP expression based on the mean fluorescence intensity. LV-shGFP significantly inhibited GFP expression by 74.7 ± 2.6% (P < 0.01), and treatment of 1, 10, or 100 IU/ml of IFN-α did not interfere with the knockdown by LV-shGFP. Shown is the mean ± SD of three independent experiments (*P < 0.01)

Fig. 4

IFN-α does not interfere with RNAi-mediated silencing of CD81. a LV-shCD81 vector containing a GFP reporter gene and shRNA targeting CD81 was used to transduce Huh7 cells. LV-GFP vector without shRNA cassette was used as control. b Three days after transfection, GFP-positive transduced cells were gated to determine CD81 expression. c Representative CD81 expression histogram of GFP-positive cells is shown. Isotype-matched control antibody staining of LV-shCD81 cells was included as negative control. d Knockdown of CD81 in Huh7 cells profoundly reduced HCV infection. Three days after LV-shCD81 transduction, Huh7 cells were exposed to JFH1-derived infectious HCV particles for 6 h. Three days after infection, the LV-shCD81 treated cells showed a clear reduction in intracellular viral RNA levels as determined by quantitative RT-PCR. e Relative CD81 expression was calculated based on mean fluorescence intensity. LV-shCD81 significantly reduced CD81 expression by 90.8 ± 8.1% (P < 0.01), compared to LV-GFP cells. LV-shCD81 retained robust gene silencing efficacy at different concentrations of IFN-α. Shown is the mean ± SD of three independent experiments (*P < 0.01)

Fig. 5

Enhanced inhibition of HCV replication by simultaneous treatment with IFN-α and RNAi. a The structure and action of LV-shNS5b on HCV replication have been shown in Fig. 1b. The combination of low-dose IFN-α (<1 IU/ml) with low-dose vector (≤10 MOI) resulted in enhanced antiviral effects. For example, combination of 1 MOI vector with 0.9 IU/ml IFN-α showed 92.1% ± 8.1 inhibition, versus 72.2% ± 9.4 with vector alone or 71.5 ± 4.1% with IFN-α alone (mean ± SD, P < 0.01). Also, with 5 MOI, a significant combinational effect was observed (P < 0.05), but at higher MOIs, inhibition of HCV replication was nearly complete, and no significant additive effect of IFN-α was observed. b LV-shIRES vector containing GFP reporter gene and shRNA targeting HCV IRES was used to transduce Huh7-ET cells. Increasing dose of LV-shIRES resulted in higher levels of transduction efficiency and inhibition of HCV replication, shown by GFP positivity and luciferase activity, respectively. c Combining low-dose IFN-α with LV-shIRES resulted in enhanced inhibition of HCV replication at each combined condition. Shown is the mean ± SD of six independent experiments (*P < 0.05)

Fig. 6

Subsequential treatment of IFN-α and RNAi reciprocally enhances inhibition of HCV replication. a Huh7-ET cells were treated with low dose (0.5 IU/ml) of IFN-α 24 h after which medium was replaced, and cells were treated a second time for an additional 86 h. Secondary treatment with IFN-α resulted in a maximum inhibition of 81.3% ± 1.3 (n = 7, P < 0.001) from t = 42 h onward. However, subsequently switching IFN-α to LV-shNS5b resulted in a significantly greater inhibition of viral replication (98.2% ± 0.2 inhibition, n = 6, P < 0.001). In Huh7-ET cells without secondary treatment, HCV replication was partially restored to approx. two third of baseline levels at 2 days after switching. b Conversely, Huh7-ET cells primarily treated with LV-shIRES were more sensitive to a secondary treatment with IFN-α than re-treatment with the same LV-shIRES vector. Secondary treatment with 0.9 IU/ml IFN-α resulted in profound inhibition of viral replication (96% ± 0.6 inhibition at t = 96 h, n = 6, P < 0.001) as compared to secondary treatment with vector (75.1% ± 3.7 inhibition, n = 6). In Huh7-ET, cells without additional treatment HCV replication was (66.4% ± 5.4 inhibition, n = 6). Overall, these findings indicate that cells treated with IFN-α are more sensitive to subsequential treatment with RNAi than re-treatment with IFN-α. Equally, cells treated with RNAi are more sensitive to subsequential treatment with IFN-α than re-treatment with RNAi