Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1
Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1
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DOI：
10.1038/mtna.2016.93
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发表时间：
2016-11-22
影响因子：
8.8
通讯作者：
Wissinger, Bernd
中科院分区：
文献类型：
--
作者：
Bonifert, Tobias;Menendez, Irene Gonzalez;Wissinger, Bernd
遗传性视神经病变（ION）是导致欧洲劳动年龄人口失明的重要原因。最近，我们报道了四个独立的家族在主要遗传性视神经病变基因OPA1中发现了深内含子突变。这些深层内含子突变通过创建隐式受体剪接位点导致OPA1前信使rna转录物的错误剪接。作为一种补救策略，我们试图通过使用2' o -甲基反义寡核苷酸（AONs）来防止突变前信使rna的错误剪接，这些AONs具有全长磷酸化主链，分别针对隐藏受体剪接位点和由深层内含子突变产生的预测新分支点。用这些AONs转染患者来源的原代成纤维细胞，可以诱导突变前信使rna以时间和浓度依赖的方式正确剪接，这是通过信息杂合变异体的焦磷酸测序检测到的。使用靶向AON的隐受体剪接位点和使用靶向AON的分支点处理显示出较强的挽救效果（类似于55%）和中度的挽救效果（类似于16%）。剪接矫正的效果在治疗后4天达到最高，但在转染后14天仍有显著效果。Western blot分析显示，OPA1蛋白的含量增加，在治疗后3天达到最大值。总之，我们提供了第一个使用合成AONs的突变特异性体外挽救策略来治疗OPA1缺乏症。
Inherited optic neuropathies (ION) present an important cause of blindness in the European working-age population. Recently we reported the discovery of four independent families with deep intronic mutations in the main inherited optic neuropathies gene OPA1. These deep intronic mutations cause mis-splicing of the OPA1 pre-messenger-RNA transcripts by creating cryptic acceptor splice sites. As a rescue strategy we sought to prevent mis-splicing of the mutant pre-messenger-RNA by applying 2'O-methyl-antisense oligonucleotides (AONs) with a full-length phosphorothioate backbone that target the cryptic acceptor splice sites and the predicted novel branch point created by the deep intronic mutations, respectively. Transfection of patient-derived primary fibroblasts with these AONs induced correct splicing of the mutant pre-messenger-RNA in a time and concentration dependent mode of action, as detected by pyrosequencing of informative heterozygous variants. The treatment showed strong rescue effects (similar to 55%) using the cryptic acceptor splice sites targeting AON and moderate rescue (similar to 16%) using the branch point targeting AON. The highest efficacy of Splice correction could be observed 4 days after treatment however, significant effects were still seen 14 days post-transfection. Western blot analysis revealed increased amounts of OPA1 protein with maximum amounts at similar to 3 days post-treatment. In summary, we provide the first mutation-specific in vitro rescue strategy for OPA1 deficiency using synthetic AONs.