Leber Hereditary Optic Neuropathy: Gene Therapy Trial

描述(申请人提供)：我们对线粒体DNA突变引起的人类疾病的理解最近发生了戏剧性的变化。这是一组影响眼睛、神经系统和心脏的无法治愈的疾病，其中一些在一个多世纪前就被临床确定为特征，但现在已知它们是一系列分子定义的疾病。我们选择从最严重的一种开始，线粒体DNA的G11778A突变导致Leber遗传性视神经病变(LHON)，这是一种以在儿童后期和成年早期导致失明而闻名的疾病。它很少或根本没有复苏的倾向。在以前的工作中，我们在确定LHON的发病机制和测试治疗方法方面取得了重大进展。首先，我们发现ND4突变细胞的ATP合成严重减少，尽管复合体I活性的轻微下降似乎不足以诱发疾病。由于没有将DNA直接导入线粒体的技术，我们克服了这一缺陷，构建了线粒体基因的“核版本”，然后通过在阅读框架上附加靶向序列(异位表达)将细胞质合成的蛋白质靶向线粒体。当被送到含有突变ND4的培养细胞中时，呼吸功能恢复了。接下来，我们为LHON创建了一个真实的动物模型。利用ND4核基因的定点突变，我们将340位氨基酸的精氨酸密码子替换为组氨酸密码子。将该基因注入小鼠视觉系统，破坏了线粒体的细胞结构，增加了活性氧，导致视神经头肿胀和诱导细胞凋亡，视网膜及其组成视神经的轴突中的神经节细胞逐渐死亡。相反，野生型人类ND4亚单位的眼睛表达似乎是安全的，这表明它可能对LHON患者的治疗有用。在这里，通过使用迄今获得的科学和临床知识，我们将开始人类视神经疾病和线粒体疾病的基因治疗之旅。我们在这项应用中的目标是测试异体挽救我们的LHON动物模型的相关性、AAV介导的ND4基因传递的安全性以及这种疗法在预防和恢复LHON患者视力损失方面的有效性。

DESCRIPTION (provided by applicant): Our understanding of human diseases caused by mutated mitochondrial DNA has recently evolved dramatically. This is a group of untreatable disorders affecting the eye, nervous system and heart, some that have been clinically characterized over a century ago, but they are now known to be a spectrum of molecularly defined diseases. We have chosen to start with one of the most severe, the G11778A mutation in mitochondrial DNA responsible for Leber Hereditary Optic Neuropathy (LHON) a disease group renowned for causing blindness in later childhood and early adulthood. It shows little or no propensity for recovery. In previous work we have made major strides towards determining the pathogenesis and testing a treatment for LHON. First, we discovered that ND4 mutant cells have a severe reduction in ATP synthesis, even though mild reductions in complex I activity appear insufficient to induce disease. Since no technology existed to introduce DNA directly into mitochondria, we overcame this deficiency in oxidative phosphorylation by constructing a "nuclear version" of the mitochondrial gene then targeted the cytoplasmically synthesized protein to the mitochondria by using a targeting sequence appended to the reading frame (allotopic expression). When delivered to cultured cells containing a mutant ND4, respiratory function was restored. Next, we created a bona fide animal model for LHON. Using site directed mutagenesis of the nuclear version of ND4 we replaced the codon for arginine to that for histidine at amino acid 340. Injection of this construct into the mouse visual system disrupted mitochondrial cytoarchitecture, elevated reactive oxygen species, induced swelling of the optic nerve head and induced apoptosis, with a progressive demise of ganglion cells in the retina and their axons comprising the optic nerve. In contrast, ocular expression of the wild-type human ND4 subunit appeared safe, suggesting that it may be useful for treatment of patients with LHON. Here by using the scientific and clinical knowledge acquired to date we will begin the journey towards genetic therapy for human optic neuropathy and mitochondrial disease. Our goal in this application is to test the relevance of allotopic rescue of our LHON animal model; the safety of AAV mediated delivery of the ND4 gene and the effectiveness of this therapy in preventing and restoring visual loss in our patients with LHON.