Inhibition of core gene of HCV 3a genotype using synthetic and vector derived siRNAs

Abstract

Background: Hepatitis C virus (HCV) is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option.

Results: The current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs) against Core gene were designed and in vitro transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed.

Conclusions: Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.

Figure 1

HCV -3a Core specific siRNAs inhibit Core expression. A) Dose dependent silencing effect of synthetic siRNA against Core gene of HCV-3a. Huh-7 cells were transfected with 0.4 μg of constructed HCV Core vector or Mock along with or without 10, 20 and 40 nM of siRNAs for 24 and 48 hrs. Cells were harvested and relative RNA determinations were carried out using semi-quantitative RT-PCR. Gene expression results are given for increasing concentrations of Csi16, Csi27, Csi151, Csi352, and Csi476 siRNAs against HCV-3a Core. Expression levels for Mock-transfected (M), HCV-3a Core expression plasmid (C), scramble siRNA (Sc), 100 bp DNA Ladder (L) and GAPDH are also shown. B) Quantitative Real Time PCR analysis of Core 3a or Mock-treated Huh-7 cells along with or without 40 nM of siRNAs for 24 and 48 hrs in comparison to Mock. Gene expression results from Real Time PCR shows that Csi16, Csi352, and Csi476 siRNAs against HCV-3a Core decrease RNA expression after 24 and 48 hrs transfection. GAPDH was used as internal control. Each independent experiment was performed having triplicate samples. The p values indicate significant differences between the connected groups. Error bars indicate mean S.D, Csi476 verses other siRNA: p* for 24 and p^ for 48 hrs C). Silencing of HCV-3a Core gene by siRNAs using specific antibodies showed reduction at protein expression level. The protein expression levels were determined by western blot analysis after 24 and 48 hrs transfection with Mock (M), HCV-3a Core expression plasmid (C) with and without HCV-3a siRNAs (Csi16, Csi27, Csi151, Csi352, and Csi476) and scramble siRNA (Sc) in Huh-7 cells.

Figure 2

Silencing effect of HCV-3a genes-specific siRNAs show a dramatic reduction of viral titer in Huh-7 cells infected with HCV-3a sera. Huh-7 cells were infected with high titer sera samples from HCV-3a patients (S3a) to establish in vitro cell culture model of HCV-3a, cells were maintained overnight at 37°C in 5% CO₂ for three days. Cells were harvested after siRNA treatment 48 hrs post transfection and intracellular HCV RNA levels were quantified by Real Time PCR. Data is expressed as mean percent viral load of non-siRNA treated samples. Nine independent experiments each with triplicate determinations were performed with different sera infected cells. Error bars indicate, mean S.D p < 0.05 verses S3a.

Figure 3

HCV-3a Core specific shRNAs inhibit mRNA expression. A) Total cellular RNA extracted from transfected Huh-7 after 24, 48 and 72 hrs post-transfection. Gene expression results from Real Time PCR showed that Csh352 and Csh476 shRNAs against HCV-3a Core decrease RNA expression using gene specific primers in comparison to Mock with GAPDH as internal control. Three independent experiments were performed having triplicate samples. Error bars indicate mean S.D, *p < 0.01. B) Silencing of HCV-3a Core gene by shRNAs using specific antibodies showed reduction at protein expression level determined by western blot analysis after 24, 48 and 72 hrs transfection with Mock (M), HCV-3a Core expression plasmid (C) with and without HCV-3a shRNAs (Csh352 and Csh476) and scramble shRNA (ScshRNA) in Huh-7 cells. Protein levels for GAPDH gene are also shown as internal control.

Figure 4

Silencing effect of HCV-3a Core-specific shRNAs show a dramatic reduction of viral titer in Huh-7 cells infected with HCV-3a sera. Huh-7 cells were infected with high titer sera samples from HCV-3a patients to establish in vitro cell culture model of HCV-3a, cells were maintained overnight at 37°C in 5% CO₂, incubation was continued for 48 hrs. Huh-7 infected cells were again plated and transfected with shRNAs against HCV-3a genes for additional 48 hrs. Cells were harvested and intracellular HCV RNA levels were quantified by Real Time PCR. Data are expressed as mean percent viral load of non-siRNA treated samples. Nine independent experiments with different sera infected cells and each with triplicate samples were performed. Error bars indicate, mean S.D p < 0.01 verses S3a.