Pharmacologic Treatment of Human Mitochondrial DNA (mtDNA) Disease

 描述（由申请人提供）：呼吸链功能障碍是一类由线粒体DNA（mtDNA）缺陷引起的破坏性和致命性神经肌肉疾病。以前认识不足，这些疾病有很高的患病率（~1：5000），目前没有通用的治疗方法。以前，研究表明，在mtDNA疾病的细胞模型中，可以利用细胞内质量控制途径来恢复线粒体功能。具体地，在异质细胞（即含有混合的细胞）中， 在正常和突变的mtDNA群体中，这是临床情况的典型），降解“缺陷”线粒体的宏观自噬途径可以通过无葡萄糖酮体饮食（“生酮疗法”）诱导，以在细胞内增加功能性线粒体的比例并降低突变的mtDNA的百分比。生酮疗法在患者中不可行，但FDA批准的药物西罗莫司/雷帕霉素已知可诱导细胞中的大自噬。雷帕霉素（rapa）治疗mtDNA疾病细胞迅速和显着增加细胞内功能性线粒体的比例，但令人惊讶的是， 突变mtDNA的百分比没有相关的变化，正如预期的那样，如果拉帕诱导大自噬。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来起作用。基于这些初步数据，将拉帕给予mtDNA疾病的小鼠模型，特别是mtDNA耗竭综合征（MDS）模型。这些MDS小鼠在编码支配mtDNA完整性的神经特异性胸苷激酶（TK 2）的核基因中具有突变，并表现出严重的进行性运动和神经症状，死亡率约为20%。14天大的在这个小鼠模型中，早期用rapa干预显著延迟了死亡率，几乎使寿命延长了一倍。本研究培训方案建议以两种方式对这些初步结果采取后续行动。具体目标1将研究在mtDNA疾病的异质细胞模型中rapa介导的功能性拯救的细胞机制。这将通过独立地在遗传和生物学上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mtDNA疾病细胞模型中的线粒体翻译和呼吸功能。具体目标2的目标是确定拉帕在挽救线粒体疾病中的最大作用以及挽救机制。首先优化治疗，然后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Tk 2缺陷改善、线粒体功能以及对下游细胞途径的影响。雷帕霉素的使用可能代表了第一种基于mtDNA的线粒体疾病的普遍治疗，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.