Towards gene therapy of mitochondrial disease

描述（由申请人提供）：广泛的遗传性人类疾病是由线粒体DNA （mtDNA）基因组和核基因组中编码的线粒体基因突变引起的，但目前没有有效的治疗这些临床毁灭性疾病的方法。我们能够改造酵母的线粒体dna，而且，我们已经开发出技术，使我们第一次能够改造哺乳动物的线粒体基因组，并将这些基因组重新引入小鼠胚胎。我们建议利用这项技术开发一种治疗弗里德赖希共济失调（FRDA）的基因疗法，FRDA是一种常染色体隐性神经退行性疾病，由核编码的线粒体蛋白frataxin缺陷引起。我们将把我们最初的努力集中在纠正与酵母中fraataxin完全丧失相关的分子缺陷上。这种表征良好的FRDA模型将使我们能够系统地评估有效表达线粒体基因组中功能齐全的fraataxin所需的一般和蛋白质特异性特征。我们将利用在项目初始阶段开发的信息和试剂来设计一组适合于纠正小鼠FRDA模型缺陷的小鼠mtDNA基因组。我们将通过将这些基因组包装在线粒体中并注射到FRDA敲除小鼠的单细胞胚胎中，来评估这些基因组补偿小鼠核frataxin基因缺失的效率。由于这种基因敲除突变导致线粒体功能丧失，因此对胚胎是致命的，我们可以很容易地通过线粒体frataxin基因部分拯救（即产生可存活的胚胎细胞）或完全拯救这种表型（即产生可存活的小鼠）的能力来测定线粒体frataxin基因的功能。因此，这个实验系统将使我们能够优化我们的线粒体转移技术和线粒体fraataxin基因结构。一旦我们完成了本申请中描述的工作，我们将处于一个很好的位置，为FRDA小鼠模型开发一种基因治疗方法，并致力于将这些疗法应用于治疗FRDA和其他人类线粒体疾病。公共卫生相关性：一系列广泛的遗传性人类疾病是由线粒体DNA （mtDNA）基因组和核基因组中编码的线粒体基因突变引起的，但目前尚无针对这些临床毁灭性疾病的有效治疗方法。本提案中描述的实验将使我们对其中一种疾病（FRDA）有更大的分子洞察力，并将使我们能够开发mtDNA工程和转移技术，这些技术将成为开发治疗这些疾病的不可或缺的工具。

DESCRIPTION (provided by applicant): A wide array of inherited human diseases are caused by mutations in our mitochondrial DNA (mtDNA) genome and in mitochondrial genes encoded in the nuclear genome, but there are currently no effective therapies for these clinically devastating diseases. We are able to engineer yeast mtDNA and, moreover, we have developed technology that allows us for the first time to engineer mammalian mitochondrial genomes and reintroduce these genomes into mouse embryos. We propose to use this technology to develop a gene therapy for Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disease caused by defects in frataxin, a nucleus-encoded mitochondrial protein. We will focus our initial efforts on correcting the molecular deficits associated with the complete loss of frataxin in yeast. This well characterized model of FRDA will enable us to systematically assess both general and protein-specific features required to efficiently express a fully functional form of frataxin from the mitochondrial genome. We will use the information and reagents developed in this initial phase of the project to engineer a set of mouse mtDNA genomes suitable for correcting deficits in a mouse model of FRDA. We will evaluate the efficiency with which these genomes compensate for the loss of the mouse nuclear frataxin gene by packaging them in mitochondria and injecting them into single cell embryos of FRDA knockout mice. Because this gene knockout mutation leads to loss of mitochondrial function and so is embryonic lethal, we can readily assay the functionality of the mitochondrial frataxin genes by their ability to either partially rescue (i.e., generate viable embryonic cells) or fully rescue this phenotype (i.e., generate viable mice). This experimental system will therefore allow us to optimize both our mitochondrial transfer technology and our mitochondrial frataxin gene constructs. Once we have completed the work described in this application we will be in an excellent position to develop a gene therapy approach for mouse models of FRDA and to work towards adapting these therapies to treating FRDA and other mitochondrial diseases in humans. PUBLIC HEALTH RELEVANCE: A wide array of inherited human diseases are caused by mutations in our mitochondrial DNA (mtDNA) genome and in mitochondrial genes encoded in the nuclear genome, but there are currently no effective therapies for these clinically devastating diseases. The experiments described in this proposal will give us both greater molecular insights into one of these diseases (FRDA) and will allow us to develop mtDNA engineering and transfer technologies that will serve as indispensable tools for developing therapies to treat these diseases.