Pharmacologic Treatment of Human Mitochondrial DNA (mtDNA) Disease

 描述(申请人提供)：呼吸链功能障碍是一类破坏性的和总是致命的神经肌肉疾病，由线粒体DNA(MtDNA)缺陷引起。以前认识不足的是，这些疾病的患病率很高(约1：5000)，目前还没有普遍可用的治疗方法。此前，研究表明，在线粒体DNA疾病的细胞模型中，可以利用细胞内质量控制通路来恢复线粒体功能。具体地说，在异质细胞中(即含有混合的 正常和突变的线粒体DNA的群体，这是典型的临床情况)，巨型自噬途径，降解“有缺陷的”线粒体可以诱导无葡萄糖的酮体饮食(“生酮疗法”)，以增加细胞内的功能线粒体的比例和减少突变的线粒体DNA的百分比。生酮疗法在患者中是不可行的，但FDA批准的药物西罗莫斯/雷帕霉素已知会在细胞中诱导巨大的自噬。雷帕霉素(RapA)治疗mtdna病细胞迅速并显著增加了细胞内功能线粒体的比例，尽管令人惊讶的是， 如果RAPA诱导巨噬细胞自噬，突变的mtDNA的百分比没有相关的变化，这是预期的。然而，观察到线粒体形态的变化，因此RAPA可能通过促进线粒体融合和细胞器间互补发挥作用。基于这些初步数据，RAPA被应用于mtDNA疾病的小鼠模型，特别是mtDNA耗竭综合征(MDS)模型。这些MDS小鼠的核基因发生突变，编码一种线粒体特异性胸苷激酶酶(TK2)，该酶控制mtDNA的完整性，并表现出严重的进行性运动和神经系统症状，死亡率约为1%。14日龄。在这种小鼠模型中，早期使用RAPA干预显著延迟了死亡率，寿命几乎翻了一番。这项研究培训计划建议通过两种方式对这些初步结果进行跟踪。具体目的1将在线粒体DNA病的异质细胞模型中探讨RAPA介导的功能挽救的细胞机制。这将通过从遗传和药物上独立抑制自噬和线粒体融合途径来实现，然后通过免疫细胞化学和创新的实时生物能量学分析来评估线粒体DNA疾病细胞模型中的线粒体翻译和呼吸功能。具体目标2的目的是确定RAPA在抢救线粒体疾病中的最大作用以及抢救机制。首先将优化治疗，然后将对治疗和对照小鼠进行酶和免疫化学分析，以评估RAPA介导的TK2缺乏症的改善、线粒体功能以及对下游细胞通路的影响。雷帕霉素的使用可能代表着对基于线粒体DNA的线粒体疾病的第一次普遍治疗，支持减轻神经系统疾病负担的使命。

 DESCRIPTION (provided by applicant): Respiratory chain dysfunction is a class of devastating and invariably fatal neuromuscular disorders caused by defects in mitochondrial DNA (mtDNA). Previously under-recognized, these disorders have a high prevalence (~1:5000) and there are currently no generalized therapies available. Previously, it was shown that in cellular models of mtDNA disease, intracellular quality control pathways could be harnessed to restore mitochondrial function. Specifically, in heteroplasmic cells (i.e. cells containing a mixed population of normal and mutated mtDNA, which is typical of the clinical situation), the macro-autophagy pathway that degrades "defective" mitochondria could be induced by a glucose-free ketone body diet ("ketogenic therapy") to intracellularly increase the proportion of functional mitochondria and decrease the percentage of mutated mtDNA. Ketogenic therapy is not feasible in patients, but the FDA-approved drug sirolimus/rapamycin is known to induce macro-autophagy in cells. Rapamycin (rapa) treatment of mtDNA disease cells rapidly and dramatically increased the intracellularly proportion of functional mitochondria, though surprisingly, there was no associated shift in percentage of mutated mtDNA, as expected if rapa induced macro-autophagy. Changes were observed in mitochondrial morphology, however, and thus rapa may work by promoting mitochondrial fusion and inter-organellar complementation. Based on these preliminary data, rapa was administered to a mouse model of mtDNA disease, specifically a model of mtDNA depletion syndrome (MDS). These MDS mice have mutations in a nuclear gene encoding a mitochondrial-specific thymidine kinase enzyme (TK2) that governs mtDNA integrity, and exhibit severe progressive motor and neurologic symptoms, with mortality by approx. 14 days old. Early intervention with rapa significantly delayed mortality in this mouse model, almost doubling lifespan. This research training program proposes to follow-up on these preliminary results in two ways. Specific Aim 1 will investigate the cellular mechanism of rapa-mediated functional rescue in heteroplasmic cell models of mtDNA disease. This will be accomplished by genetically and pharmacologically inhibiting the autophagic and mitochondrial fusion pathways, independently, and then assessing mitochondrial translation and respiratory function in cell models of mtDNA disease through immunocytochemistry and innovative real-time bioenergetics analysis. The goal of Specific Aim 2 is to determine the maximum effect of rapa in rescuing mitochondrial disease, as well as the rescue mechanism. Therapy will first be optimized, and then enzymatic and immunochemical analysis of treated and control mice will be performed to assess rapa-mediated amelioration of Tk2 deficiency, mitochondrial function, and effects on downstream cellular pathways. Use of rapamycin may represent the first generalized treatment for mtDNA-based mitochondrial disorders, supporting the mission of reducing the burden neurologic disease.