title: RNA interference in vitro and in vivo using DsiRNA targeting the nucleocapsid N mRNA of human metapneumovirus.
abstract: Human metapneumovirus causes respiratory diseases with outcomes that can be severe in children, the immunocompromised, and the elderly. Synthetic small interfering RNAs (siRNAs) that silence targeted genes can be used as therapeutic agents. Currently, there is no specific therapy for hMPV. In this study, we designed Dicer-substrate siRNAs (DsiRNAs) that target metapneumovirus sequences on the mRNAs of the N, P, and L genes. In vitro, six DsiRNAs were shown to inhibit virus replication using cell proliferation tests. Of those, the DsiRNA that targets the most conserved mRNA sequence was then resynthesized in Evader™ format with heavy 2'-O-methyl modification of the guide strand. In a murine model, the prophylactic administration of this Evader™ DsiRNA was effective at partially inhibiting viral replication of hMPV (13×10(3) vs. 29×10(3)PFU/g of lung; p<0.01), which was not the case for the control, a mismatched DsiRNA. Inhibition was achieved without inducing cytokines or off-target effects. Moreover, the specificity of the siRNA mechanism of action was demonstrated in vitro and in vivo using 5'-RACE methodology. This in vivo approach of using a DsiRNA against hMPV is an important step in the development of synthetic siRNA as a therapeutic agent for this virus.
authors.affiliation: Laboratoire de Virologie, CHU Dijon, 2 Rue Angélique Ducoudray, 21070 Dijon cedex, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Darniot
authors.forename: Verena
authors.initials: V
authors.lastname: Schildgen
authors.forename: Oliver
authors.initials: O
authors.lastname: Schildgen
authors.forename: Brian
authors.initials: B
authors.lastname: Sproat
authors.forename: Michael
authors.initials: M
authors.lastname: Kleines
authors.forename: Vanessa
authors.initials: V
authors.lastname: Ditt
authors.forename: Cécile
authors.initials: C
authors.lastname: Pitoiset
authors.forename: Pierre
authors.initials: P
authors.lastname: Pothier
authors.forename: Catherine
authors.initials: C
authors.lastname: Manoha
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005260:Female; D006801:Humans; D029121:Metapneumovirus / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D018184:Paramyxoviridae Infections / Q000188:drug therapy* / Q000201:enzymology / Q000821:virology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use*; D043244:Ribonuclease III / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D014764:Viral Proteins; D043244:Ribonuclease III