Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs.
Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs.
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DOI：
10.1093/nar/gkw676
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发表时间：
2016-09-19
影响因子：
14.9
通讯作者：
Minczuk M
中科院分区：
文献类型：
--
作者：
Gammage PA;Gaude E;Van Haute L;Rebelo-Guiomar P;Jackson CB;Rorbach J;Pekalski ML;Robinson AJ;Charpentier M;Concordet JP;Frezza C;Minczuk M
线粒体疾病通常与线粒体DNA（mtDNA）的突变有关。在大多数情况下，突变型和野生型mtDNA共存，导致异质性。突变mtDNA的选择性消除和随之而来的野生型mtDNA的富集，可以挽救异质细胞中的病理表型。利用线粒体靶向的锌指核酸酶（mtZFN）通过位点特异性DNA切割导致突变mtDNA的降解。在这里，我们描述了我们以前的mtZFN为基础的方法，以靶向mtDNA的大幅增强，允许接近完整的方向转移的mtDNA异质性，无论是通过迭代治疗或通过精细控制的表达mtZFN，这限制了脱靶催化和不希望的mtDNA拷贝数耗尽。为了证明这种改进的方法的实用性，我们产生了一个等基因分布的异质性细胞与可变的mtDNA突变水平，从相同的父母来源，没有克隆选择。对这些群体的分析表明，在携带m.8993T>G突变mtDNA水平降低的细胞中，代谢特征发生改变，与神经病、共济失调和色素性视网膜炎（NARP）相关。我们的结论是，基于mtZFN的方法提供了在基础研究中mtDNA异质性操纵的手段，并可能提供一种策略，在选定的线粒体疾病的治疗干预。
Mitochondrial diseases are frequently associated with mutations in mitochondrial DNA (mtDNA). In most cases, mutant and wild-type mtDNAs coexist, resulting in heteroplasmy. The selective elimination of mutant mtDNA, and consequent enrichment of wild-type mtDNA, can rescue pathological phenotypes in heteroplasmic cells. Use of the mitochondrially targeted zinc finger-nuclease (mtZFN) results in degradation of mutant mtDNA through site-specific DNA cleavage. Here, we describe a substantial enhancement of our previous mtZFN-based approaches to targeting mtDNA, allowing near-complete directional shifts of mtDNA heteroplasmy, either by iterative treatment or through finely controlled expression of mtZFN, which limits off-target catalysis and undesired mtDNA copy number depletion. To demonstrate the utility of this improved approach, we generated an isogenic distribution of heteroplasmic cells with variable mtDNA mutant level from the same parental source without clonal selection. Analysis of these populations demonstrated an altered metabolic signature in cells harbouring decreased levels of mutant m.8993T>G mtDNA, associated with neuropathy, ataxia, and retinitis pigmentosa (NARP). We conclude that mtZFN-based approaches offer means for mtDNA heteroplasmy manipulation in basic research, and may provide a strategy for therapeutic intervention in selected mitochondrial diseases.
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影响因子：
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作者：
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作者：
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通讯作者：
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