RNA interference in vitro and in vivo using DsiRNA targeting the nucleocapsid N mRNA of human metapneumovirus

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• 作者：Magali Darniot^a ; ¹ ; Verena Schildgen^b ; ¹ ; ² ; ^{schildgeno@kliniken-koeln.de} ; Oliver Schildgen^b ; ¹ ; ² ; Brian Sproat^c ; ¹ ; ³ ; ^{fa205401@skynet.be} ; Michael Kleines^d ; ¹ ; ⁴ ; ^{michael.kleines@i-med.ac.at} ; Vanessa Ditt^b ; ¹ ; ² ; C&eacute ; cile Pitoiset^a ; ¹ ; Pierre Pothier^a ; ¹ ; Catherine Manoha^a ; ¹ ; ^{catherine.manoha@chu-dijon.fr}
• 关键词：hMPV ; human metapneumovirus ; N ; nucleoprotein ; RNAi ; RNA interference ; D ; dicer ; siRNA ; small interfering RNA ; RISC ; RNA-induced silencing complex ; TLR ; Toll-like receptor ; IFN-伪 ; interferon-伪 ; IL-6 ; interleukin-6 ; TNF-伪 ; tumor necrosis factor-伪 ; i.n.
• 刊名：Antiviral Research
• 出版年：2012
• 期刊代码：7_01663542
• 类别：imm
• 出版时间：March, 2012
• 卷：93
• 期：3
• 页码：364-373
• 文件大小：728 K

摘要

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鈩?format with heavy 2鈥?O-methyl modification of the guide strand. In a murine model, the prophylactic administration of this Evader鈩?DsiRNA was effective at partially inhibiting viral replication of hMPV (13 脳 10³ vs. 29 脳 10³ 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鈥?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.