Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: Implications for the therapeutic development of RNAi
Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: Implications for the therapeutic development of RNAi
复制标题
DOI：
10.1073/pnas.0801775105
复制
发表时间：
2008-04-15
影响因子：
11.1
通讯作者：
Davidson, Beverly L.
中科院分区：
文献类型：
--
作者：
McBride, Jodi L.;Boudreau, Ryan L.;Davidson, Beverly L.
亨廷顿氏病是一种致命的显性神经退行性疾病，由编码亨廷顿蛋白的亨廷顿氏病基因外显子1的聚谷氨酰胺重复扩增引起。我们和其他人已经证明，RNAi是一种治疗HID的候选疗法，因为表达针对突变的人类HID转基因的抑制性rna可以改善HID小鼠模型中的神经病理学和行为缺陷。在这里，我们开发了靶向人类HID和小鼠HID同源（HDh） mrna中的保守序列的shrna，在敲入小鼠HID模型中进行临床前测试。我们在体外筛选了35种shrna，随后将重点缩小到3种候选的体内测试。出乎意料的是，两种活性shrna在小鼠纹状体中诱导了显著的神经毒性，尽管这三种shrna都将HDh mRNA的表达降低到相似的水平。此外，含有错配的对照shRNA也会引起毒性，尽管它没有降低HDh mRNA的表达。有趣的是，与无毒shRNA相比，有毒shRNA产生了更高的反义RNA水平。这些结果表明，从shRNA表达系统中出现的强大水平的反义rna在小鼠大脑中可能存在问题。重要的是，当在shrna背景下具有毒性的序列被置于人工microRNA （miRNA）表达系统中时，神经毒性的分子和神经病理读数显着减弱，而不影响小鼠HDh沉默的效果。因此，基于mirna的方法可能为在大脑中表达抑制性rna提供更合适的生物学工具，这对RNAi的基础生物学和治疗应用的发展至关重要。
Huntington's disease (HID) is a fatal, dominant neurodegenerative disease caused by a polyglutamine repeat expansion in exon 1 of the HID gene, which encodes the huntingtin protein. We and others have shown that RNAi is a candidate therapy for HID because expression of inhibitory RNAs targeting mutant human HID transgenes improved neuropathology and behavioral deficits in HID mouse models. Here, we developed shRNAs targeting conserved sequences in human HID and mouse HID homolog (HDh) mRNAs to initiate preclinical testing in a knockin mouse model of HID. We screened 35 shRNAs in vitro and subsequently narrowed our focus to three candidates for in vivo testing. Unexpectedly, two active shRNAs induced significant neurotoxicity in mouse striatum, although HDh mRNA expression was reduced to similar levels by all three. Additionally, a control shRNA containing mismatches also induced toxicity, although it did not reduce HDh mRNA expression. Interestingly, the toxic shRNAs generated higher antisense RNA levels, compared with the nontoxic shRNA. These results demonstrate that the robust levels of antisense RNAs emerging from shRNA expression systems can be problematic in the mouse brain. Importantly, when sequences that were toxic in the context of shRNAs were placed into artificial microRNA (miRNA) expression systems, molecular and neuropathological readouts of neurotoxicity were significantly attenuated without compromising mouse HDh silencing efficacy. Thus, miRNA-based approaches may provide more appropriate biological tools for expressing inhibitory RNAs in the brain, the implications of which are crucial to the development of RNAi for both basic biological and therapeutic applications.