RNA interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy

Abstract

We describe the design and characterization of a potent human respiratory syncytial virus (RSV) nucleocapsid gene-specific small interfering RNA (siRNA), ALN-RSV01. In in vitro RSV plaque assays, ALN-RSV01 showed a 50% inhibitory concentration of 0.7 nM. Sequence analysis of primary isolates of RSV showed that the siRNA target site was absolutely conserved in 89/95 isolates, and ALN-RSV01 demonstrated activity against all isolates, including those with single-mismatch mutations. In vivo, intranasal dosing of ALN-RSV01 in a BALB/c mouse model resulted in potent antiviral efficacy, with 2.5- to 3.0-log-unit reductions in RSV lung concentrations being achieved when ALN-RSV01 was administered prophylactically or therapeutically in both single-dose and multidose regimens. The specificity of ALN-RSV01 was demonstrated in vivo by using mismatch controls; and the absence of an immune stimulatory mechanism was demonstrated by showing that nonspecific siRNAs that induce alpha interferon and tumor necrosis factor alpha lack antiviral efficacy, while a chemically modified form of ALN-RSV01 lacking measurable immunostimulatory capacity retained full activity in vivo. Furthermore, an RNA interference mechanism of action was demonstrated by the capture of the site-specific cleavage product of the RSV mRNA via rapid amplification of cDNA ends both in vitro and in vivo. These studies lay a solid foundation for the further investigation of ALN-RSV01 as a novel therapeutic antiviral agent for clinical use by humans.

FIG. 1.

Structure of ALN-RSV01 duplex and IC₅₀ of ALN-RSV01 in vitro. (A) ALN-RSV01 is a synthetic double-stranded RNA oligonucleotide formed by hybridization of two partially complementary single-stranded RNAs in which the 3′ ends are capped with two thymidine units. (B) Vero cells in 24-well plates were transfected with decreasing concentrations of ALN-RSV01, followed by infection with 200 to 300 PFU of RSV A2. At 5 days postinfection, the cells were fixed, immunostained, and counted. The activity of ALN-RSV01 was plotted as a percentage of that of PBS, and the IC₅₀ was measured with XLFit software. Error bars represent standard deviations.

FIG. 2.

Genotype analysis of RSV primary isolates. Maximum-likelihood phylogenetic trees for RSV serotype A (A) and RSV serotype B (B) strains on the basis of analysis of the RSV G-protein gene. Bootstrap values of ≥70% (1,000 replicates) are shown at the corresponding nodes. Circles, isolates whose ALN-RSV01 target sites were analyzed.

FIG. 3.

In vitro inhibition of primary RSV isolates by ALN-RSV01. Vero cells in 24-well plates were transfected with decreasing concentrations of ALN-RSV01, followed by infection with 200 to 300 PFU of RSV primary isolates. At day 5 postinfection, the cells were fixed, immunostained, and counted. The activity of ALN-RSV01 was plotted as a percentage of that of PBS.

FIG. 4.

In vivo activity of ALN-RSV01 following administration of single and multiple doses in BALB/c mice. In each panel, each group represents five animals, error bars represent standard deviations, and the threshold of detection of the assay is 1.7 log₁₀ PFU/g. (A) ALN-RSV01 in vivo dose-response curve. BALB/c mice were treated i.n. with ALN-RSV01 at increasing concentrations (25 μg, 50 μg, or 100 μg), negative control siRNA AL-DP-1730 (1730; 100 μg), or PBS 4 h prior to infection with 1 × 10⁶ PFU of RSV A2. The lungs were harvested, and virus was quantified by a standard immunostaining plaque assay and plotted as log₁₀ PFU/g lung. (B) ALN-RSV01 multidose study. BALB/c mice were treated i.n. with ALN-RSV01 or mismatch siRNA (AL-DP-1730) at either 40 μg, 80 μg, or 120 μg (single-dose treatment) or 40 μg (multiple-dose, daily treatment). The lungs were harvested, and virus was quantified by an immunostaining plaque assay on day 5. −4, 4 h prior to infection; D1, day 1 postinfection; D2, day 2 postinfection; D3, day 3 postinfection. (C) ALN-RSV01 same-day multidose study. BALB/c mice were treated i.n. with ALN-RSV01 or mismatch siRNA (1730) at either 40 μg, 60 μg, 80 μg, or 120 μg for the single-dose groups at day 1 or 2 after RSV infection or 40 μg two or three times daily for the multidose groups at day 1 or 2 after RSV infection. The lungs were harvested, and virus was quantified by an immunostaining plaque assay.

FIG. 5.

ALN-RSV01 is a modest stimulator of IFN-α and TNF-α in vitro. Transfection reagent alone (Txn) or siRNAs (ALN-RSV01 [RSV01] or mismatch-positive controls siRNA AL-DP-7296 [7296] and siRNA AL-DP-5048 [5048]) were transfected into PBMCs and assayed by ELISA for cytokine induction at 24 h posttransfection. (A) IFN-α induction; (B) TNF-α induction.

FIG. 6.

TNF-α- and IFN-α-stimulatory mismatched siRNAs do not modulate RSV in vivo. (A) Transfection reagent alone (Txn) or siRNAs (AL-DP-5048 [5048] and AL-DP-1730 [1730]) transfected into PBMCs were assayed for TNF-α stimulation at 24 h posttransfection. (B) siRNAs (AL-DP-5048 [5048] and AL-DP-2153 [2153]) transfected into PBMCs were assayed for IFN-α stimulation at 24 h posttransfection. (C) Lung virus concentrations from mice dosed i.n. with RSV at day 0 and with RSV-specific siRNAs (ALN-RSV01) or mismatch control immunostimulatory siRNAs (AL-DP-2153 [2153] and AL-DP-1730 [1730]) at 4 h before inoculation. Lung RSV concentrations were measured by an immunostaining plaque assay at day 5 postinfection.

FIG. 7.

Chemically modified ALN-RSV01 (AL-DP-16571) is immunologically silent, while it maintains antiviral activity. (A) Transfection reagent alone (Txn), AL-DP-16570 (16570), or positive control siRNAs AL-DP-5048 (5048) and AL-DP-7296 (7296) were transfected into PBMCs and assayed for IFN-α stimulation at 24 h posttransfection. (B) AL-DP-16570 or positive control siRNAs AL-DP-5048 and AL-DP-7296 were transfected into PBMCs and assayed for TNF-α stimulation at 24 h postinfection. (C) Lung virus concentrations from mice dosed i.n. with RSV at day 0 and with ALN-RSV01 (RSV01), AL-DP-16570 (16570), or a mismatched control at the indicated concentrations at 4 h before inoculation. Lung RSV concentrations were measured by an immunostaining plaque assay at day 5 postinfection.

FIG. 8.

ALN-RSV01 viral inhibition is mediated by RNAi in vivo. Shown is a schematic representation of the 5′ RACE assay used to demonstrate the generation of the site-specific cleavage product. Boxed are the results of sequence analysis of individual clones from PCR amplification of cDNAs generated from linker-adapted RSV N-gene mRNA isolated from an in vivo viral inhibition assay in which mice were inoculated with RSV at day 0 and treated with ALN-RSV01, AL-DP-2153 (Neg Control), or PBS at day 3, followed by lung homogenization and evaluation by RACE at day 5 postinfection.