RNA interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy.
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.
Alvarez
Rene
R
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, USA.
Elbashir
Sayda
S
Borland
Todd
T
Toudjarska
Ivanka
I
Hadwiger
Philipp
P
John
Mathias
M
Roehl
Ingo
I
Morskaya
Svetlana Shulga
SS
Martinello
Rick
R
Kahn
Jeffrey
J
Van Ranst
Mark
M
Tripp
Ralph A
RA
DeVincenzo
John P
JP
Pandey
Rajendra
R
Maier
Martin
M
Nechev
Lubomir
L
Manoharan
Muthiah
M
Kotelianski
Victor
V
Meyers
Rachel
R
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
D000998:Antiviral Agents; D016898:Interferon-alpha; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha