Leber Hereditary Optic Neuropathy: Gene Therapy Trial

描述（由申请人提供）：我们对线粒体 DNA 突变引起的人类疾病的理解最近发生了巨大的变化。这是一组影响眼睛、神经系统和心脏的无法治疗的疾病，其中一些疾病在一个多世纪前就已经有了临床特征，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，即线粒体 DNA 中的 G11778A 突变，该突变导致莱伯遗传性视神经病 (LHON)，这种疾病因导致儿童后期和成年早期失明而闻名。它显示出很少或没有恢复的倾向。在之前的工作中，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，尽管复合物 I 活性的轻微减少似乎不足以诱发疾病。由于不存在将 DNA 直接引入线粒体的技术，我们通过构建线粒体基因的“核版本”，然后使用附加到阅读框（同位素表达）的靶向序列将细胞质合成的蛋白质靶向线粒体，从而克服了氧化磷酸化的缺陷。当递送至含有突变体 ND4 的培养细胞时，呼吸功能得到恢复。接下来，我们为 LHON 创建了一个真正的动物模型。使用ND4核版本的定点诱变，我们将氨基酸340处的精氨酸密码子替换为组氨酸密码子。将该构建体注射到小鼠视觉系统中会破坏线粒体细胞结构，升高活性氧，诱导视神经乳头肿胀并诱导细胞凋亡，视网膜中的神经节细胞及其构成视神经的轴突逐渐死亡。相比之下，野生型人 ND4 亚基的眼部表达似乎是安全的，这表明它可能有助于治疗 LHON 患者。在这里，通过利用迄今为止获得的科学和临床知识，我们将开始人类视神经病变和线粒体疾病的基因治疗之旅。我们在此应用中的目标是测试我们的 LHON 动物模型的同位素救援的相关性； AAV 介导的 ND4 基因传递的安全性以及该疗法在预防和恢复 LHON 患者视力丧失方面的有效性。

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.