Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.
Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.
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DOI：
10.1038/srep30377
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发表时间：
2016-07-27
影响因子：
4.6
通讯作者：
Kugimiya A
中科院分区：
文献类型：
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作者：
Kasuya T;Hori S;Watanabe A;Nakajima M;Gahara Y;Rokushima M;Yanagimoto T;Kugimiya A
Gapmer反义寡核苷酸在体内有效地切割靶RNA，被认为是有前途的治疗剂。特别是，用锁核酸（LNA）修饰的缺口聚体显示出强大的敲除活性;然而，它们也会引起肝毒性副作用。为了开发安全有效的gapmer药物，需要更深入地了解肝毒性的机制。在这里，我们调查了来自LNA修饰的gapmer的肝毒性的原因。gapmer的gap区域的化学修饰完全抑制了敲低活性和肝毒性，表明肝毒性的根本原因与细胞内gapmer活性有关。肝核糖核酸酶H1（RNaseH 1）的基因沉默可以催化缺口体介导的RNA敲除，强烈抑制肝毒性作用。小干扰RNA（siRNA）介导的靶mRNA敲低没有导致任何肝毒性作用，而靶向与siRNA相同的mRNA位置的gapmer显示急性毒性。微阵列分析显示，含有类似于gapmer靶标的序列的几种前mRNA也被敲低。这些结果表明LNA gapmer的肝毒性是由RNAseH 1活性引起的，推测是由于核内RNA的脱靶切割。
Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer’s gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei.