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

Alvarez, Rene; Elbashir, Sayda; Borland, Todd; Toudjarska, Ivanka; Hadwiger, Philipp; John, Mathias; Roehl, Ingo; Morskaya, Svetlana Shulga; Martinello, Rick; Kahn, Jeffrey; Van Ranst, Mark; Tripp, Ralph A; DeVincenzo, John P; Pandey, Rajendra; Maier, Martin; Nechev, Lubomir; Manoharan, Muthiah; Kotelianski, Victor; Meyers, Rachel

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.