Gene therapy for mitochondrial disease by delivering restriction endonuclease Smal into mitochondria
Gene therapy for mitochondrial disease by delivering restriction endonuclease Smal into mitochondria
复制标题
DOI：
10.1159/000064726
复制
发表时间：
2002-01-01
影响因子：
11
通讯作者：
Yagi, K
中科院分区：
文献类型：
--
作者：
Tanaka, M;Borgeld, HJ;Yagi, K
限制性内切核酸酶Smal已用于诊断神经源性肌无力、共济失调和视网膜色素变性疾病或Leigh病，其由Mt 8993 T->G突变引起，该突变导致Leu 156 Arg置换，其阻断F0 F1-ATP酶的亚基a的质子易位活性。我们的最终目标是将Smal应用于这种疾病的基因治疗，因为突变型mtDNA（mtDNA）与野生型mtDNA共存（异质性），并且因为只有突变型mtDNA而不是野生型mtDNA被酶选择性限制。为了这个目的，我们瞬时表达Smal基因融合的线粒体靶向序列携带突变mtDNA的胞质杂种。在这里，我们证明，线粒体靶向的Smal酶表现出特定的消除突变mtDNA。这种消除之后，再通过野生型mtDNA进行重新增殖，导致恢复正常的细胞内ATP水平和正常的线粒体膜电位。此外，在体内电穿孔的质粒表达靶向EcoRI诱导仓鼠骨骼肌细胞色素c氧化酶活性的降低，而不引起细胞核的退行性变化。将限制性内切酶递送到线粒体中是一种用于特殊形式的线粒体疾病的基因治疗的新策略。版权所有（C）2002国家科学理事会，中华民国和S. Karger AG，巴塞尔。
The restriction endonuclease Smal has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993T-->G mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F0F1-ATPase. Our ultimate goal is to apply Smal to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA) coexists with the wild-type mtDNA (heteroplasmy), and because only the mutant mtDNA, but not the wild-type mtDNA, is selectively restricted by the enzyme. For this purpose, we transiently expressed the Smal gene fused to a mitochondrial targeting sequence in cybrids carrying the mutant mtDNA. Here, we demonstrate that mitochondria targeted by the Smal enzyme showed specific elimination of the mutant mtDNA. This elimination was followed with repopulation by the wild-type mtDNA, resulting in restoration of both the normal intracellular ATP level and normal mitochondrial membrane potential. Furthermore, in vivo electroporation of the plasmids expressing mitochondrion-targeted EcoRI induced a decrease in cytochrome c oxidase activity in hamster skeletal muscles while causing no degenerative changes in nuclei. Delivery of restriction enzymes into mitochondria is a novel strategy for gene therapy of a special form of mitochondrial diseases. Copyright (C) 2002 National Science Council, ROC and S. Karger AG, Basel.