Department of Anatomy I, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.
Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.
Sci Rep. 2017 Nov 14;7(1):15557. doi: 10.1038/s41598-017-15871-y.
Induced pluripotent stem cells (iPSCs) are suitable for studying mitochondrial diseases caused by mitochondrial DNA (mtDNA) mutations. Here, we generated iPSCs from a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) with the m.13513G>A mutation. The patient's dermal fibroblasts were reprogrammed, and we established two iPSC clones with and without mutant mtDNA. Furthermore, we tried to decrease mutant mtDNA level in iPSCs using transcription activator-like effector nucleases (TALENs). We originally engineered platinum TALENs, which were transported into mitochondria, recognized the mtDNA sequence including the m.13513 position, and preferentially cleaved G13513A mutant mtDNA (G13513A-mpTALEN). The m.13513G>A heteroplasmy level in MELAS-iPSCs was decreased in the short term by transduction of G13513A-mpTALEN. Our data demonstrate that this mtDNA-targeted nuclease would be a powerful tool for changing the heteroplasmy level in heteroplasmic iPSCs, which could contribute to elucidation of the pathological mechanisms of mitochondrial diseases caused by mtDNA mutations.
诱导多能干细胞（iPSCs）适合研究由线粒体 DNA（mtDNA）突变引起的线粒体疾病。在这里，我们从一位患有线粒体肌病、脑病、乳酸酸中毒和卒中样发作（MELAS）的患者中生成了具有 m.13513G>A 突变的 iPSCs。患者的皮肤成纤维细胞被重编程，我们建立了两个具有和不具有突变 mtDNA 的 iPSC 克隆。此外，我们尝试使用转录激活因子样效应物核酸酶（TALENs）来降低 iPSCs 中的突变 mtDNA 水平。我们最初设计了铂 TALENs，这些 TALENs被运送到线粒体中，识别包括 m.13513 位置的 mtDNA 序列，并优先切割 G13513A 突变 mtDNA（G13513A-mpTALEN）。通过转导 G13513A-mpTALEN，MELAS-iPSCs 中的 m.13513G>A 异质性水平在短期内降低。我们的数据表明，这种靶向 mtDNA 的核酸酶将成为改变异质 iPSCs 中异质性水平的有力工具，这有助于阐明由 mtDNA 突变引起的线粒体疾病的病理机制。
