Screening for potent HCV IRES-targeting siRNA by siRNA tiling experiments.

<p>(A) The proposed secondary structure of HCV IRES. The IRES region spanning nts 277–343 (shown in gray) was targeted by siRNAs. The target site of the selected potent anti-HCV siRNA siIE22 is shown in blue. The base-pairings in the proposed pseudoknot (PK) structures (PKs 1 and 2) are shown in green. (B) siRNA sequences tiled over HCV IRES. The underlined sequence represents a mapped druggable region (nts 313–343) where the targets of selected potent siRNAs were enriched. (C) Anti-HCV activity of HCV IRES-targeting siRNAs in Huh7 cells transfected with Rluc-JFH1 (top panel), an HCV replicon encoding the Rluc reporter. The Rluc gene was fused in frame to the DNA sequence encoding 17 N-terminal amino acid residues of the HCV core protein. Huh7 cells were electroporated with the Rluc-JFH1 <i>in vitro</i> RNA transcript and pGL3 plasmid used for normalization of transfection efficiency. After 24 h, the cells were transfected with each of the IRES-specific siRNAs or a scrambled (Sc) siRNA (50 nM). At 48 h post-transfection, luciferase activity was measured. (D) Huh7 cells harboring an HCV subgenomic replicon RNA (R-1) were transfected with each of the IRES-specific siRNAs or Sc siRNA (10 nM each). At 48 h post-transfection, HCV RNA levels were quantified by real-time qRT-PCR. *, <i>P <</i> 0.01. (E and F) Dose-dependent inhibition of HCV-replication by siIE22 was assessed in R-1 cells, as described in (D). HCV genome copy number and HCV proteins (NS5A and NS5B) levels were analyzed by qRT-PCR (E) and western blotting (F), respectively.</p

Inhibition of HCV IRES-mediated translation by systemically delivered LNP-formulated siIE22.

<p>(A and B) Schematic diagram of siIE22 LNP (A) and LNP particle size analysis (B). (C) Experimental schedule and schematic representation of the pDual-IRES plasmid. The pDual-IRES plasmid was hydrodynamically injected through the tail vein of BALB/c mice (n = 4 per group). After 1 h, mice were iv injected with siIE22 LNP at a dose of 1 mg/kg body weight. The Fluc expression level in the liver was determined 16 h after the injection. Luciferase activity is reported as RLU per mg protein. *, <i>P <</i> 0.01. (D) BALB/c mice (n = 4 per group) were iv injected with indicated siRNAs (1 mg/kg body weight) complexed with ND98 or formulated with LNP. Poly(I:C) (1 mg/kg) complexed with ND98, formulated with LNP, or free form (each in 170 μl) was administered. PBS or LNP vesicles alone were used as control treatments. Two hours later, serum IFN-α levels were quantified by ELISA. The dotted line indicates the detection limit of the assay (15 pg/ml). (E) hPMBCs grown in 96-well plates were transfected with indicated siRNA at 10 nM concentration or with 1 μg/well poly(I:C) using the lipidoid ND98 or stimulated by a direct addition of 50 μg/ml poly(I:C) to the medium. After 16 h, cell culture supernatants from stimulated cells were analyzed for IFN-α by ELISA. Data shown are from one of the two independent experiments with similar results. ND, non-detectable. (F) HEK293 cells were transfected with the luciferase expressing plasmids (IFNβ-pGL3 and pRL-TK) for the IFN-β promoter activity assay. After 6 h, cells were transfected with 100 nM siIE22 or scrambled (Sc) siRNA, or 1 μg/ml poly(I:C). After 8 h, cells were harvested for dual luciferase assays. Fluc activity was normalized to Rluc activity from the pRL-TK plasmid. Normalized luciferase activity (Fluc/Rluc) of mock-treated cells was defined as 100. Data are presented as the mean ± SD of six measurements from two independent experiments.</p

Anti-HCV efficacy of chemically modified siIE22 derivatives.

<p>(A) Non-modified siIE22 (2 μM) was incubated in 45% human plasma for the indicated time periods. RNA extracted from each sample was resolved by electrophoresis on a denaturing 15% polyacrylamide gel and subjected to northern blotting (NB) analysis for detection of siIE22 guide-strand. The Phosphorimager image shown is from one representative experiment of three independent experiments with similar results. Densitometric analysis of siIE22 guide strand signal was done using a Phosphorimager. Relative intensity of signals was plotted using SigmaPlot to estimate siIE22 guide strand half-life. Relative signal (% of signal at time 0) is shown below a representative blot. (B) Sequences of modified guide and passenger strands of siIE22 derivatives used in this study. Modified residues are shown in green or blue. “s”, phosphorothioate linkage. (C) Plasma stability of a set of the selected siIE22 derivatives was evaluated as in (A). (D) Anti-HCV activity of the selected modified siIE22 (1 nM) was evaluated in R-1 cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146710#pone.0146710.g001" target="_blank">Fig 1D</a>. *, <i>P <</i> 0.01. (E) Analysis of half-life of the gs_PS1 siIE22 as in (A).</p

Anti-HCV efficacy of gs_PS1 siIE22 LNP in a mouse model for HCV replication.

<p>(A) HCV genome in the HCV-replicating Huh7 (+ HCV) or Huh7 (- HCV) sc xenograft was detected by northern blotting. An <i>in vitro</i> transcribed HCV RNA genome was used as a size marker. The 28S rRNA detected by ethidium bromide staining is shown as a loading control. (B) Immunostaining for HCV viral proteins (E2 and NS5B) in the xenograft at 4 weeks post-xenografting. DAPI, nuclear staining. Scale bar, 10 μm. (C) The NOD-SCID mice (n = 3) carrying HCV-replicating Huh7 xenograft were treated with gs_PS1 siIE22 LNP at a dose of 1 mg/kg body weight via tail vein injection. Shown are relative serum HCV RNA titers at the indicated time points. *, <i>P</i> < 0.01. (D) Relative serum HCV RNA titers in the mice treated with LNP-formulated Sc siRNA (Sc LNP) or gs_PS1 siIE22 LNP (1 mg/kg) once in every three days for 4 times. The data were generated from two independent experiments with a total of 6 mice per group. Each differently colored diamond represents an individual mouse. *, <i>P</i> < 0.01.</p

Analysis of anti-HCV potency and RNAi activity of various siRNAs targeting the IRES subdomain IIIf.

<p>(A) Passenger strand sequences of siIE22 and six siRNAs sharing their targets with siIE22 in the IRES subdomain IIIf. (B) Evaluation of anti-HCV efficacy of a set of selected siRNAs (100 pM each) using an HCV replicon expressing an Rluc reporter. Luciferase activity at 48 h post-siRNA treatment is shown. Sc, scrambled siRNA. (C) Antiviral efficacy of gs_PS1 siIE22 and siIE318_27 in R-1 cells harboring an HCV subgenomic replicon. (D) <i>In vitro</i> target cleavage assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146710#pone.0146710.g002" target="_blank">Fig 2A</a>. P, 5′ ³²P-radiolabeled 31-nt long HCV IRES probe; CP, cleaved probe. (E) Comparison of antiviral activity of indicated siRNAs targeting the region shared with siIE22 in R-1 cells. In (B), (C), and (E), *, <i>P <</i> 0.05; **, <i>P <</i> 0.01.</p

Target RNA cleavage activity and serum stability of the selected IRES-targeting siRNAs.

<p>(A) <i>In vitro</i> cleavage assays were performed with 5′ ³²P-radiolabeled 31-nts long HCV IRES (spanning nts 313–343) using Ago2 complexes immunoprecipitated from HeLa cells transfected with each indicated siRNA (20 nM). Sc, scrambled siRNA. (B) RNA target cleavage assay was performed with 5′ ³²P-radiolabeled HCV IRES substrate (spanning nts 1–487) using a recombinant human Ago2 protein and guide strand of indicated siRNAs. As a negative control, siIE22 passenger-strand was used. RNA samples were analyzed by electrophoresis on a denaturing polyacrylamide denaturing gel followed by autoradiography. (C) Serum stability of siRNAs was assessed by incubating siRNAs in 45% plasma for the indicated time periods and subsequent quantification of remained intact siRNA guide-strand by Phosphorimager analysis of northern blots. (D) The cleavage assay was performed as described in (B). Radioactivity of cleaved products was quantified by Phosphorimager analysis and shown below the autoradiogram (mean ± SD of three independent experiments).</p