Guy John, Qi Xiaoping, Pallotti Francesco, Schon Eric A, Manfredi Giovanni, Carelli Valerio, Martinuzzi Andrea, Hauswirth William W, Lewin Alfred S
Department of Ophthalmology, Neuro-Opthalmology Service, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Ann Neurol. 2002 Nov;52(5):534-42. doi: 10.1002/ana.10354.
A G to A transition at nucleotide 11778 in the ND4 subunit gene of complex I was the first point mutation in the mitochondrial genome linked to a human disease. It causes Leber Hereditary Optic Neuropathy, a disorder with oxidative phosphorylation deficiency. To overcome this defect, we made a synthetic ND4 subunit compatible with the "universal" genetic code and imported it into mitochondria by adding a mitochondrial targeting sequence. For detection we added a FLAG tag. This gene was inserted in an adeno-associated viral vector. The ND4FLAG protein was imported into the mitochondria of cybrids harboring the G11778A mutation, where it increased their survival rate threefold, under restrictive conditions that forced the cells to rely predominantly on oxidative phosphorylation to produce ATP. Since assays of complex I activity were normal in G11778A cybrids we focused on changes in ATP synthesis using complex I substrates. The G11778A cybrids showed a 60% reduction in the rate of ATP synthesis. Relative to mock-transfected G11778A cybrids, complemented G11778A cybrids showed a threefold increase in ATP synthesis, to a level indistinguishable from that in cybrids containing normal mitochondrial DNA. Restoration of respiration by allotopic expression opens the door for gene therapy of Leber Hereditary Optic Neuropathy.
复合体I的ND4亚基基因中第11778位核苷酸处的G到A转换是线粒体基因组中与人类疾病相关的首个点突变。它会导致Leber遗传性视神经病变，这是一种伴有氧化磷酸化缺陷的疾病。为了克服这一缺陷，我们制备了一种与“通用”遗传密码兼容的合成ND4亚基，并通过添加线粒体靶向序列将其导入线粒体。为了进行检测，我们添加了一个FLAG标签。该基因被插入腺相关病毒载体中。ND4FLAG蛋白被导入携带G11778A突变的胞质杂种的线粒体中，在迫使细胞主要依靠氧化磷酸化来产生ATP的限制性条件下，它使细胞存活率提高了三倍。由于G11778A胞质杂种中复合体I活性检测结果正常，我们将重点放在使用复合体I底物时ATP合成的变化上。G11778A胞质杂种的ATP合成速率降低了60%。相对于mock转染的G11778A胞质杂种，互补的G11778A胞质杂种的ATP合成增加了三倍，达到与含有正常线粒体DNA的胞质杂种无法区分的水平。通过异位表达恢复呼吸作用为Leber遗传性视神经病变的基因治疗打开了大门。
