RNAi-Mediated Silencing of the Respiratory Syncytial Virus Nucleocapsid Defines a Potent Anti-Viral Strategy.
(2009)
Journal - Antimicrobial agents and chemotherapy
Abstract :
Here we describe the design and characterization of a potent RSV nucleocapsid gene-specific siRNA, ALN-RSV01. In in vitro RSV plaque assays, ALN-RSV01 showed an IC50 of 0.7 nM. Across sequence analysis of primary isolates of RSV, the siRNA target site was absolutely conserved in 89/95 isolates, and ALNRSV01 demonstrated anti-viral 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 anti-viral efficacy with 2.5-3.0 log reductions in RSV lung concentrations when administered prophylactically or therapeutically, in both single dose and multidose regimens. The specificity of ALN-RSV01 was demonstrated in vivo using mismatch controls, and the absence of an immune stimulatory mechanism was demonstrated by showing that nonspecific siRNAs that induce IFN-alpha and TNF-alpha lack anti-viral efficacy while a chemically modified form of ALN-RSV01 lacking measurable immunostimulatory capacity, retained full in vivo activity. Further, an RNAi mechanism of action was demonstrated by the capture of the site specific cleavage product of the RSV mRNA via rapid amplification of cDNA ends (RACE) both in vitro and in vivo. These studies lay a solid foundation for the further investigation of ALN-RSV01 as a novel anti-viral therapeutic for human clinical use.