体内 siRNA 递送的定量评估。
Quantitative evaluation of siRNA delivery in vivo.
机构信息
Department of RNA Therapeutics, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
出版信息
RNA. 2010 Dec;16(12):2553-63. doi: 10.1261/rna.2255810. Epub 2010 Oct 12.
Abstract
Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.
摘要
有效的小干扰 RNA（siRNA）介导的治疗需要将 siRNA 递送至细胞 RNA 诱导的沉默复合物（RISC）中。目前，该关键递送步骤的定量信息是根据组织中基因沉默和 siRNA 摄取的效果推断出来的。在这里，我们报告了一种在啮齿动物和猴子中直接定量 RISC 中 siRNA 的方法。该方法通过从组织裂解物中特异性免疫沉淀 RISC，并通过茎环 PCR 定量免疫沉淀物中的小 RNA 来实现。该方法预计与递送载体和靶标无关，是无标记的，并且对于临床前动物研究的通量是可接受的。我们通过整合这些方法来表征脂质制剂的 siRNA，并获得了关于 siRNA 组织积累、RISC 加载和基因沉默的定量视角。所描述的方法学对于沉默机制的研究、siRNA 治疗的开发和临床试验设计具有实用价值。
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