title: 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.
authors.affiliation: Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, USA.
authors.forename: Rene
authors.initials: R
authors.lastname: Alvarez
authors.forename: Sayda
authors.initials: S
authors.lastname: Elbashir
authors.forename: Todd
authors.initials: T
authors.lastname: Borland
authors.forename: Ivanka
authors.initials: I
authors.lastname: Toudjarska
authors.forename: Philipp
authors.initials: P
authors.lastname: Hadwiger
authors.forename: Mathias
authors.initials: M
authors.lastname: John
authors.forename: Ingo
authors.initials: I
authors.lastname: Roehl
authors.forename: Svetlana Shulga
authors.initials: SS
authors.lastname: Morskaya
authors.forename: Rick
authors.initials: R
authors.lastname: Martinello
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Kahn
authors.forename: Mark
authors.initials: M
authors.lastname: Van Ranst
authors.forename: Ralph A
authors.initials: RA
authors.lastname: Tripp
authors.forename: John P
authors.initials: JP
authors.lastname: DeVincenzo
authors.forename: Rajendra
authors.initials: R
authors.lastname: Pandey
authors.forename: Martin
authors.initials: M
authors.lastname: Maier
authors.forename: Lubomir
authors.initials: L
authors.lastname: Nechev
authors.forename: Muthiah
authors.initials: M
authors.lastname: Manoharan
authors.forename: Victor
authors.initials: V
authors.lastname: Kotelianski
authors.forename: Rachel
authors.initials: R
authors.lastname: Meyers
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D006224:Cricetinae; D005260:Female; D005838:Genotype; D006801:Humans; D016898:Interferon-alpha / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D019251:Nucleocapsid / Q000235:genetics*; D010802:Phylogeny; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000378:metabolism; D018357:Respiratory Syncytial Virus Infections / Q000188:drug therapy / Q000235:genetics / Q000821:virology*; D018113:Respiratory Syncytial Virus, Human / Q000145:classification / Q000187:drug effects* / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D016898:Interferon-alpha; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha