Artificial MicroRNAs as siRNA Shuttles: Improved Safety as Compared to shRNAs In vitro and In vivo
Artificial MicroRNAs as siRNA Shuttles: Improved Safety as Compared to shRNAs In vitro and In vivo
复制标题
DOI：
10.1038/mt.2008.231
复制
发表时间：
2009-01-01
影响因子：
12.4
通讯作者：
Davidson, Beverly L.
中科院分区：
文献类型：
--
作者：
Boudreau, Ryan L.;Martins, Ines;Davidson, Beverly L.
RNA干扰（RNAi）为人类疾病的治疗提供了一种很有前途的方法。然而，最近报道的数据表明，短发夹rna （shRNAs）可能导致细胞毒性，这需要进一步研究使用RNAi载体的安全性。先前，在比较基于发夹的RNAi载体时，我们注意到shrna高表达并产生大量未经加工的前体，而人工microrna （mirna）表达水平较低且加工效率较高。我们假设未加工的shrna来自内源性RNAi机制的饱和，这可能对细胞造成负担。在本研究中，我们通过评估shrna和人工mirna对mirna加工和功能的相对影响来验证这一假设。在竞争分析中，shrna破坏了miRNA的生物发生和功能，而人工miRNA即使在与shrna一样有效的沉默剂量下也避免了这种干扰。接下来，我们比较了这些载体在小鼠小脑中的安全性，发现shrna引起浦肯野细胞神经毒性。相比之下，人工miRNA表达耐受性良好，导致浦肯野细胞中有效的靶基因沉默。这些发现，连同来自小鼠纹状体的早期研究数据，表明基于mirna的平台更适合于哺乳动物大脑的治疗性沉默。
RNA interference (RNAi) provides a promising therapeutic approach to human diseases. However, data from recent reports demonstrate that short-hairpin RNAs (shRNAs) may cause cellular toxicity, and this warrants further investigation of the safety of using RNAi vectors. Earlier, in comparing hairpin-based RNAi vectors, we noted that shRNAs are highly expressed and yield an abundance of unprocessed precursors, whereas artificial microRNAs (miRNAs) are expressed at lower levels and are processed efficiently. We hypothesized that unprocessed shRNAs arise from the saturation of endogenous RNAi machinery, which poses likely a burden to cells. In this study, we tested that hypothesis by assessing the relative effects of shRNAs and artificial miRNAs on the processing and function of miRNAs. In competition assays, shRNAs disrupted miRNA biogenesis and function, whereas artificial miRNAs avoided this interference even when dosed to silence as effectively as shRNAs. We next compared the safety of these vectors in mouse cerebella, and found that shRNAs cause Purkinje cell neurotoxicity. By contrast, artificial miRNA expression was well tolerated, resulting in effective target gene silencing in Purkinje cells. These findings, together with data from earlier work in mouse striata, suggest that miRNA-based platforms are better suited for therapeutic silencing in the mammalian brain.