THERAP APPROACHES OF CELL MODELS /MITOCHONDRIAL DISEASE

在上一个资助期间，我们成功地开发了一种基因方法来拯救 使用一种称为“同位素表达”的策略，发现由于线粒体 DNA (mtDNA) 编码的 ATPase 6 突变导致母系遗传 Leigh 综合征 (MILS) 细胞模型中 ATP 合成不足。我们还开始致力于开发治​​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）处理含有 MILS 突变的异质细胞，导致异质性快速稳定地转变为有利于野生型 mtDNA，同时线粒体功能得到改善。重要的是，我们现在已经证明，这种类型的“异质转移”策略可以应用于其他呼吸复合体中的线粒体缺陷，包括卡恩斯-塞尔综合征（KSS）中的线粒体缺陷，其特征是 mtDNA（δ-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）作为唯一碳 源，以便选择功能并显着减少复制细胞系统（即细胞质杂合[cybrid]细胞）中 delta-mtDNA 的数量。我们现在建议通过三种方式跟进这种有希望的治疗方法。首先，我们要问生酮培养基是否可以选择含有其他致病性 mtDNA 的细胞中的野生型功能和 mtDNA 突变，以便了解这种治疗策略可以推广到什么程度，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）以及低水平葡萄糖存在的情况下发挥作用，以便更接近地模拟临床情况。其次，我们会问是否可以改变终末分化、非复制模型系统（即肌管）中的异质性，以便解决生酮培养基中针对突变 mtDNA 的细胞间与细胞内选择问题。最后，我们将在 KSS 细胞中进行荧光原位杂交以确定 更加机械化的方式，在葡萄糖（“丰富”培养基）和酮（“选择性”培养基）中生长期间，线粒体DNA如何在分裂（即杂种）和非分裂（即肌管）细胞中分离。这些实验旨在为我们观察酮介导的针对突变 mtDNA 的选择提供更机制的基础，作为使用基于生酮的方案治疗 mtDNA 异质致病性突变患者的前奏。

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.