In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes

Abstract

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that mediates the sequence-specific degradation of targeted RNA and thus provides a tremendous opportunity for development of oligonucleotide-based drugs. Here, we report on the design and validation of small interfering RNAs (siRNAs) targeting highly conserved regions of the hepatitis C virus (HCV) genome. To aim for therapeutic applications by optimizing the RNAi efficacy and reducing potential side effects, we considered different factors such as target RNA variations, thermodynamics and accessibility of the siRNA and target RNA, and off-target effects. This aim was achieved using an in silico design and selection protocol complemented by an automated MysiRNA-Designer pipeline. The protocol included the design and filtration of siRNAs targeting highly conserved and accessible regions within the HCV internal ribosome entry site, and adjacent core sequences of the viral genome with high-ranking efficacy scores. Off-target analysis excluded siRNAs with potential binding to human mRNAs. Under this strict selection process, two siRNAs (HCV353 and HCV258) were selected based on their predicted high specificity and potency. These siRNAs were tested for antiviral efficacy in HCV genotype 1 and 2 replicon cell lines. Both in silico-designed siRNAs efficiently inhibited HCV RNA replication, even at low concentrations and for short exposure times (24h); they also exceeded the antiviral potencies of reference siRNAs targeting HCV. Furthermore, HCV353 and HCV258 siRNAs also inhibited replication of patient-derived HCV genotype 4 isolates in infected Huh-7 cells. Prolonged treatment of HCV replicon cells with HCV353 did not result in the appearance of escape mutant viruses. Taken together, these results reveal the accuracy and strength of our integrated siRNA design and selection protocols. These protocols could be used to design highly potent and specific RNAi-based therapeutic oligonucleotide interventions.

Fig 1. Scheme of HCV IRES and binding sites of siRNAs used in this study.

The HCV IRES and the adjacent core sequence with the binding sites of the selected in silico-designed and reference siRNAs (HCV258, HCV 321, HCV353, and HCV360) used in this study are presented here. The first nucleotide of each individual siRNA binding to the IRES is underlined. Three of the most critical IRES loops (IIId, IIIf, and IV) have been targeted by HCV353 and HCV258 siRNAs. The 5′NTR scheme was modified with permission from the Nature Publishing Group (Lukavsky et al. [11]).

Fig 2. Flowchart of steps and methodology for in silico siRNA design.

Steps 1–3: HCV IRES sequences of different genotypes were selected and aligned. All possible siRNAs were designed and sorted according to the parameters, as depicted. The candidates were scored according to first- and second-generation algorithms, and the 70% and 90% experimental threshold inhibition scores were determined. Step 4: Multi-stage filtering of siRNAs based on threshold scores, off-targets, palindromes, and repeat-motifs was performed, followed by selection of siRNAs with terminal ends mapped to a crucial IRES loop. Step 5: Additional off-target seed-region matches were filtered, and siRNAs with optimal thermodynamic properties were selected.

Fig 3. Dose-dependent antiviral effects of HCV IRES-specific siRNAs on the replication of subgenomic HCV replicons expressing a NS5A-GFP fusion protein.

(A) Representative confocal images of GFP replicon cells after transfection with HCV-specific siRNAs. Replicon cells were PFA-fixed, and stained with Hoechst 33432 stain. Images of 0.1 and 50 nM siRNA transfections at 72 h post-transfection (p.t.) are shown (ImageXpress Ultra: 20x magnification). Overlaid images: cell nuclei (blue) and HCV RNA replicating cells (green). GFP replicon cells: (B) Huh-7 Con1, (C) Huh-7 JFH-1, and (D) HuH6 JFH-1 replicon cells were plated and transfected with 0.1–50 nM siRNAs targeting the viral genome and a scrambled control. At 72 h p.t., confocal images of GFP replicon cells were acquired and analyzed using an ImageXpress Ultra microscope and MetaXpress software, respectively. Results were normalized to the level of GFP-positive cells of the scrambled siRNA transfection control, which was set to 100%. The threshold of GFP was defined by treatment of replicon cells with an HCV replication inhibitor, which was set to 100% inhibition (data not shown). Fluorescence intensity above this threshold was considered to indicate active HCV replication. Data are presented as the mean ± SEM values for four wells measured in quadruplicate in two independent experiments (N = 32). Black columns indicate HCV RNA replication (GFP-positive cells); grey columns indicate cell viability (total cell number of Hoechst 33432 stained cell nuclei). The numbers on the bars indicate the residual percentage of GFP-positive cells. Asterisks indicate that the mean values are significantly different between samples (*p<0.05; **p<0.01; ***p<0.001).

Fig 4. Dose-dependent antiviral effects of HCV IRES-specific siRNAs on the replication of subgenomic HCV replicons expressing Firefly luciferase.

Luciferase replicon cells: (A) Huh-7 Con1 and (B) Huh-7 JFH-1 replicon cells were plated and transfected with 0.1–50 nM siRNAs. At 72 h post-transfection (p.t.), cells were harvested for luciferase activity measurement. Firefly luciferase activity (HCV RNA replication) and cellular ATP content (cell viability) of the scrambled siRNA transfection were set to 100% and used to normalize the relative luciferase activities of all other siRNA-transfected cells. Data are presented as the mean ± SEM values of four wells measured in duplicate in two independent experiments (N = 24). Black columns indicate HCV RNA replication; grey columns indicate cell viability. The numbers on the bars indicate the residual percentage of luciferase activity. Asterisks indicate that the mean values are significantly different between samples (*p<0.05; **p<0.01; ***p<0.001).

Fig 5. Time-dependent antiviral effects of HCV IRES-specific siRNAs on the replication of subgenomic HCV replicons expressing Firefly luciferase.

Luciferase replicon cells: (A) Huh-7 Con1 and (B) Huh-7 JFH-1 replicon cells were plated and transfected with 0.1 nM siRNAs and analyzed as described in Fig 4. At the indicated time points (24, 48, 72 h post-transfection), cells were lysed and luciferase activities were measured. Relative luciferase activities were normalized to that of the scrambled siRNA transfection control.

Fig 6. Time-dependent antiviral effects of HCV IRES-specific siRNAs on patient-derived HCV genotype 4 isolates.

Huh-7 cells infected with patient-derived genotype 4 isolates, transfected with HCV353, HCV258, and negative control siRNAs were harvested at the indicated time points (24, 48, 72 h post-transfection) and HCV genomes determined using qRT-PCR analysis. Black and white columns indicate HCV-specific siRNA and negative control siRNAs, respectively. X-axes and y-axes depict the time post-transfection and HCV genomes, respectively. (A) HCV353 and (B) HCV258 siRNA inhibition of HCV genotype 4. Data are presented as the mean ± SEM values of two wells measured in triplicate.

Fig 7. Curing of HCV replicon cells with IRES-specific siRNAs.

(A) Subgenomic HCV JFH-1 replicon cells expressing an NS5A-GFP fusion protein were treated in the absence of G418 twice per week with 50 nM of siRNAs, either individually (scramble, HCV321 or HCV353) or in combination (50 nM HCV321 and 50 nM HCV353) for 8 weeks as depicted. HCV RNA replication was determined twice per week using GFP expression. (B) siRNA transfection was discontinued after 8 weeks, replicon cells were treated with 500 μg/mL G418 for 4 weeks, and resistant cell clones were stained using crystal violet.