Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways
Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways
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DOI：
10.1038/nature04791
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发表时间：
2006-05-25
期刊：
影响因子：
64.8
通讯作者：
Kay, Mark A.
中科院分区：
文献类型：
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作者：
Grimm, Dirk;Streetz, Konrad L.;Kay, Mark A.
RNA干扰（RNAi）是一种普遍的和进化上保守的转录后基因沉默现象，通过序列特异性mRNA降解，由小的双链RNA触发1，2。由于这种机制可以通过从非病毒和病毒载体表达靶互补短发夹RNA（shRNA）在体内有效地诱导，因此RNAi对于功能基因组学和人类治疗是有吸引力的（3，4）。在这里，我们系统地研究了成年小鼠肝脏中持续高水平shRNA表达的长期影响。静脉输注后，所有肝细胞中的稳健shRNA表达均通过基于含AAV-DNA的腺相关病毒8型（AAV 8）的优化shRNA递送载体实现。对49种不同的AAV/shRNA载体的评估显示，36种载体导致剂量依赖性肝损伤，23种载体最终导致死亡。发病与肝脏来源的microRNA（miRNAs）的下调有关，表明后者可能与shRNA竞争限制各种小RNA加工所需的细胞因子。在体外和体内的shRNA转染研究表明，一个这样的因素，共享的shRNA/miRNA途径，并容易饱和，是核karyopherin exportin-5。我们的发现对未来动物和人类基于RNAi的策略具有根本性的影响，因为控制细胞内shRNA表达水平将是必要的。然而，内源性小RNA途径过饱和的风险可以通过优化shRNA剂量和序列来最小化，正如我们在体内针对人B型肝炎病毒的持续性和治疗性RNAi的报告所示。
RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs1,2. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics(3,4). Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.