Reverse Mitochondrial Genetics Enabled by Blast

 描述(申请人提供)：线粒体是哺乳动物细胞必不可少的细胞器。它们为生物合成过程产生能量和三氯乙酸循环代谢产物，调节细胞内钙离子流量和铁-S簇的合成，并启动细胞凋亡。为了组装线粒体，需要由线粒体(MtDNA)和核(GDNA)基因组编码的蛋白质和RNA。在人类中，组成线粒体的1000种蛋白质中只有13种编码在母系遗传的~16.6kb的mtDNA中，但这13种蛋白质是使细胞呼吸得以实现的电子传输链的重要组成部分。线粒体DNA突变会影响这13种蛋白质的翻译、组装或功能，导致&gt；200被称为线粒体疾病综合征，这种疾病会影响大脑、肌肉或心脏等高能器官，并经常导致过早死亡。不幸的是，目前还没有有效的治疗方法或支持措施来治疗线粒体疾病。主要希望是最终纠正或补偿有害的mtDNA突变。然而，与相对容易地改变gDNA序列相比，存在用于改变mtDNA的几乎完整的场块。许多实验室都在试图开发线粒体反向遗传学，在这种遗传学中，改变线粒体DNA会产生表型，用于研究。然而，目前的方法是低效的、控制不佳的随机过程，往往带有对细胞来源材料的伦理担忧。几个实验室已经成功地分离、修改并在体外将改变的mtDNA重新引入线粒体，并显示出转录活性，强烈表明组装成称为类核的核酸-蛋白质聚集体。然而，没有办法将这些mtDNA工程线粒体重新引入细胞进行功能和系统范围的研究。在这里，我们建议启用线粒体反向遗传学，并提供一种纠正破坏性mtDNA突变的初步方法。在长期的合作中，Chiou和Teitell实验室发明了一种光热纳米母细胞，可以将天然或经工程改造的线粒体转移到哺乳动物细胞中，并修复细胞呼吸的缺陷。然而，我们目前的纳米棒所需的技术、缓慢的速度和整体系统的大小导致了许多失败的实验，并阻碍了这种方法的广泛采用。为了克服这些抑制问题，我们提出了三个具体的研究目标。在目标1中，我们将产生一个高通量、紧凑、微流控的平台，用于大规模并行的线粒体传递，我们称之为BLAST。在目标2中，我们将部署BLAST来生成或纠正特定的mtDNA突变，这些突变通过自然线粒体转移导致3种人类疾病综合征。在目标3中，我们将改变mtDNA并利用BLAST通过将体外修饰的线粒体转移回细胞来产生杂交细胞系，以彻底评估功能活性，包括一直无法执行的全系统碳追踪研究。结合起来，我们的工程学和分子生物学跨学科方法将使线粒体DNA的有针对性的改变成为基础、基础研究和未来线粒体医学翻译应用的开端。

 DESCRIPTION (provided by applicant): Mitochondria are essential organelles for mammalian cells. They produce energy (ATP) and TCA cycle metabolites for biosynthetic processes, regulate intracellular Ca2+ flux and Fe-S cluster synthesis, and initiate apoptosis. To assemble a mitochondrion, proteins and RNAs encoded by both the mitochondrial (mtDNA) and nuclear (gDNA) genomes are required. In humans, only 13 of >1,000 proteins that comprise a mitochondrion are encoded within maternally inherited ~16.6kb mtDNA, but these 13 proteins are essential components of the electron transport chain that enables cellular respiration. Mutations in mtDNA affecting the translation, assembly, or function of these 13 proteins results in > 200 named mitochondrial disease syndromes that affect high energy organs such as the brain, muscle, or heart and often result in early death. Unfortunately, there are no effective therapies or supportive measures for mtDNA diseases. The main hope is to eventually correct or compensate for deleterious mtDNA mutations. However, an almost complete field block exists for altering mtDNA, in contrast to comparatively ready access for altering gDNA sequences. Numerous labs are trying to develop mitochondrial reverse genetics, in which altering mtDNAs generates phenotypes for study. However, current approaches are inefficient, poorly controlled stochastic processes often with ethical concerns over the cell source materials. Several labs have managed to isolate, modify, and re-introduce altered mtDNA back into mitochondria in vitro and shown transcriptional activity, strongly suggesting assembly into nucleic acid-protein aggregates called nucleoids. However, there is no way to reintroduce these mtDNA engineered mitochondria back into cells for functional, system-wide studies. Here, we propose to enable mitochondrial reverse genetics and provide an initial approach for correcting devastating mtDNA mutations. In a longstanding collaboration, the Chiou and Teitell labs invented a photothermal nanoblade that can transfer native or engineered mitochondria into mammalian cells and rescue defects in cellular respiration. However, the skill required, slow speed, and bulk system size of our current nanoblade leads to many failed experiments and precludes wide adoption of this approach. To overcome these inhibitory issues, we propose 3 specific study aims. In Aim 1, we will generate a high throughput, compact, microfluidic platform for massively parallel mitochondrial delivery that we call BLAST. In Aim 2, we will deploy BLAST to generate or correct specific mtDNA mutations that cause 3 human disease syndromes with native mitochondrial transfers. And in Aim 3, we will alter mtDNA and utilize BLAST to generate hybrid cell lines by transfer of in vitro modified mitochondria back into cells for thorough evaluation of functional activity, including system-wide carbon tracing studies that have been impossible to perform. Combined, our engineering and molecular biology cross-disciplinary approaches will enable the targeted alteration of mtDNA for both fundamental, basic studies and the beginnings of future translational applications in mitochondrial medicine.