MitoTALEN: A General Approach to Reduce Mutant mtDNA Loads and Restore Oxidative Phosphorylation Function in Mitochondrial Diseases
MitoTALEN: A General Approach to Reduce Mutant mtDNA Loads and Restore Oxidative Phosphorylation Function in Mitochondrial Diseases
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DOI：
10.1038/mt.2015.126
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发表时间：
2015-10-01
影响因子：
12.4
通讯作者：
Moraes, Carlos T.
中科院分区：
文献类型：
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作者：
Hashimoto, Masami;Bacman, Sandra R.;Moraes, Carlos T.
我们设计了线粒体靶向转录激活子样效应核酸酶（mitoTALENs）来切割线粒体DNA （mtDNA）中的特定序列，目的是消除携带致病性点突变的mtDNA。为了测试该方法的普遍性，我们设计了mitoTALENs，针对与线粒体疾病相关的两种相对常见的致病性mtDNA点突变：与肌阵挛性癫痫伴红纤维粗糙（MERRF）相关的m.8344A>G tRNA（Lys）基因突变和与MELAS/Leigh综合征相关的m.13513G>A ND5突变。分别转染线粒体线粒体dna异质突变的线粒体杂交细胞，并在不同时间后对其进行分析。MitoTALENs有效地降低了各自细胞系中靶向致病性mtdna的水平。功能分析表明，转染mitoTALEN后，携带异质突变mtDNA的细胞能够恢复呼吸能力和氧化磷酸化酶的活性。为了在mtDNA低复杂性的背景下改进设计，我们设计了针对MERRF m.8344A >g突变的更短版本的mitoTALEN。这些较短的mitoTALENs也消除了突变的mtDNA。这些尺寸的减小将提高我们将这些大序列包装成病毒载体的能力，使这些遗传工具的使用更接近临床试验。
We have designed mitochondrially targeted transcription activator-like effector nucleases or mitoTALENs to cleave specific sequences in the mitochondrial DNA (mtDNA) with the goal of eliminating mtDNA carrying pathogenic point mutations. To test the generality of the approach, we designed mitoTALENs to target two relatively common pathogenic mtDNA point mutations associated with mitochondrial diseases: the m.8344A>G tRNA(Lys) gene mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) and the m.13513G>A ND5 mutation associated with MELAS/Leigh syndrome. Transmitochondrial cybrid cells harbouring the respective heteroplasmic mtDNA mutations were transfected with the respective mitoTALEN and analyzed after different time periods. MitoTALENs efficiently reduced the levels of the targeted pathogenic mtDNAs in the respective cell lines. Functional assays showed that cells with heteroplasmic mutant mtDNA were able to recover respiratory capacity and oxidative phosphorylation enzymes activity after transfection with the mitoTALEN. To improve the design in the context of the low complexity of mtDNA, we designed shorter versions of the mitoTALEN specific for the MERRF m.8344A>G mutation. These shorter mitoTALENs also eliminated the mutant mtDNA. These reductions in size will improve our ability to package these large sequences into viral vectors, bringing the use of these genetic tools closer to clinical trials.