THERAP APPROACHES OF CELL MODELS /MITOCHONDRIAL DISEASE

细胞模型/线粒体疾病的治疗方法

• 批准号: 6859044
• 负责人: ERIC A. SCHON
• 金额: $ 31.85万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6859044
• 关键词: aminoacid; central nervous system disorders; chemotherapy; diet therapy; disease /disorder model; drug interactions; fatty acids; fluorescent in situ hybridization; gene mutation; genetic disorder; glucose; human tissue; ketones; lactic acidosis; mental retardation; mitochondrial DNA; mitochondrial disease /disorder; myoblasts; myotubes; neuropharmacology; nutrition related tag; point mutation; retinitis pigmentosa; therapy design /development; tissue /cell culture

In the last granting period, we were successful in developing a genetic approach to rescue the deficiency in ATP synthesis in cellular models of maternally-inherited Leigh syndrome (MILS) due to mutations in mitochondrial DNA (mtDNA)-encoded ATPase 6, using a strategy called "allotopic expression." We also began work on developing a pharmacological approach to treat these disorders, based on our finding that treatment of heteroplasmic cells containing the MILS mutation with an ATPase-specific inhibitor (oligomycin) in medium containing galactose resulted in a rapid and stable shift in heteroplasmy in favor of wild-type mtDNAs, with a concomitant improvement in mitochondrial function. Importantly, we have now shown that this type of heteroplasmic shifting" strategy can be applied to mitochondrial deficiencies in other respiratory complexes, including those in Kearns-Sayre syndrome (KSS), which is characterized by large-scale deletions of mtDNA (delta-mtDNAs). Furthermore, instead of using the relatively toxic galactose/oligomycin medium, we were able to use a relatively non-toxic medum containing ketone bodies (i.e. acetoacetate and/or beta-hydroxybutyrate) as the sole carbon source in order to select for function and reduce significantly the amount of delta-mtDNAs in a replicating cell system (i.e. cytoplasmic hybrid [cybrid] cells). We now propose to follow up on this promising approach to therapy in three ways. First, we will ask if ketogenic media can select for wild-type function, and mtDNAs, in cells containing other pathogenic mtDNA mutations, in order to see the degree to which this treatment strategy can be generalized, and will also ask if ketogenic selection can work in the presence of other substrates (fatty acids, amino acids) as well as in the presence of low levels of glucose, so as to mimic the clinical situation more closely. Second, we will ask if we can shift heteroplasmy in a terminally-differentiated, non-replicating, model system, namely myotubes, in order to nail down the issue of inter- vs -intra-cellular selection against mutated mtDNAs in ketogenic medium. Finally, we will perform fluorescent in situ hybridization in KSS cells to determine in a more mechanistic fashion how mtDNAs segregate in both dividing (i.e. cybrid) and non-dividing (i.e. myotube) cells, during growth in both glucose ("rich" medium) and in ketones ("selective" medium). These experiments are designed to provide a more mechanistic underpinning for our observation of ketone-mediated selection against mutated mtDNAs, as a prelude to using a ketogenic-based protocol to treat patients with heteroplasmic pathogenic mutations in mtDNA.

在上一个资助期内，我们成功地开发了一种基因方法， 利用一种称为“异位表达”的策略，在母系遗传的Leigh综合征（MILS）细胞模型中，由于线粒体DNA（mtDNA）编码的ATP酶6突变而导致ATP合成缺乏。“我们还开始研究开发一种治疗这些疾病的药理学方法，基于我们的发现，在含有半乳糖的培养基中用ATP酶特异性抑制剂（寡霉素）治疗含有MILS突变的异质性细胞，导致异质性的快速和稳定转变，有利于野生型mtDNA，伴随着线粒体功能的改善。重要的是，我们现在已经表明，这种类型的异质性转移”策略可以应用于其他呼吸复合体中的线粒体缺陷，包括Kearns-Sayre综合征（KSS）中的线粒体缺陷，其特征是线粒体DNA（delta-mtDNA）的大规模缺失。此外，不使用 相对有毒的半乳糖/寡霉素培养基，我们能够使用相对无毒的培养基， 含有酮体（即乙酰乙酸酯和/或β-羟基丁酸酯）作为唯一的碳 来源，以便选择功能并显著减少复制细胞系统（即细胞质杂种[cybrid]细胞）中delta-mtDNA的量。我们现在建议从三个方面跟进这种有希望的治疗方法。首先，我们将询问生酮培养基是否可以在含有其他致病性mtDNA的细胞中选择野生型功能和mtDNA 突变，以便了解这种治疗策略可以推广的程度，并且还将询问生酮选择是否可以在其他底物（脂肪酸、氨基酸）存在下以及在低水平葡萄糖存在下起作用，以便更接近地模拟临床情况。第二，我们将询问我们是否可以在终末分化的、非复制的模型系统（即肌管）中改变异质性，以便确定生酮培养基中针对突变的mtDNA的细胞间与细胞内选择的问题。最后，我们将在KSS细胞中进行荧光原位杂交，以确定在一个 更机械的方式mtDNA如何在分裂（即胞质杂种）和非分裂（即肌管）细胞中分离，在葡萄糖（“富含”培养基）和酮（“选择性”培养基）中生长。这些实验旨在为我们观察酮介导的抗突变mtDNA的选择提供更多的机制基础，作为使用基于生酮的方案治疗mtDNA异质性致病突变患者的前奏。