RNA Interference-Mediated Silencing of the Respiratory Syncytial Virus Nucleocapsid Defines a Potent Antiviral Strategy
RNA Interference-Mediated Silencing of the Respiratory Syncytial Virus Nucleocapsid Defines a Potent Antiviral Strategy
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DOI：
10.1128/aac.00014-09
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发表时间：
2009-09-01
影响因子：
4.9
通讯作者：
Meyers, Rachel
中科院分区：
文献类型：
--
作者：
Alvarez, Rene;Elbashir, Sayda;Meyers, Rachel
我们描述了一种有效的人类呼吸道合胞病毒(RSV)核衣壳基因特异性小干扰RNA(SiRNA)的设计和特性，ALN-RSV01。在体外RSV空斑实验中，ALN-RSV01的半数抑制浓度为0.7 nM。对RSV原代分离株的序列分析表明，89/95分离株的siRNA靶点是绝对保守的，ALN-RSV01对所有分离株，包括单错配突变的分离株都显示了活性。在体内，在BALB/c小鼠模型中，鼻腔注射ALN-RSV01可产生强大的抗病毒效果，当ALN-RSV01预防或治疗时，单剂和多剂量方案均可使RSV肺浓度降低2.5-3.0log单位。ALN-RSV01的特异性在体内通过错配对照得到证实；免疫刺激机制的缺乏通过表明诱导α干扰素和肿瘤坏死因子α的非特异性siRNAs缺乏抗病毒效果，而缺乏可测量的免疫刺激能力的化学修饰形式的ALN-RSV01在体内保留了全部活性。此外，RNA干扰的作用机制是通过在体外和体内快速扩增cDNA端来捕获RSV mRNA的定点切割产物。这些研究为进一步研究ALN-RSV01作为一种新型的临床治疗抗病毒药物奠定了坚实的基础。
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.