Reverse Mitochondrial Genetics Enabled by Blast

Blast 实现反向线粒体遗传学

• 批准号: 9444321
• 负责人: Pei-Yu Chiou
• 金额: $ 3.19万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9444321
• 关键词: Adoption; Affect; Aging; Alpha Cell; Alzheimer' s Disease; Apoptosis; Back; Bacteria; Blast Cell; Bone Marrow; Brain; Caliber; Carbon; Cardiovascular Diseases; Cell Line; Cell Nucleus; Cell Respiration; Cell membrane; Cell physiology; Cells; Cellular Metabolic Process; Cessation of life; Child; Citric Acid Cycle; Collaborations; Complement; Complex; Coupled; DNA Sequence Alteration; Defect; Diabetes Mellitus; Disease; Electron Transport; Energy Metabolism; Engineering; Enzymes; Ethics; Evaluation; Family; Film; Future; Genetic; Genetic Transcription; Genome; Genomic DNA; Heart; Human; Hybrid Cells; Impairment; In Vitro; Individual; Inherited; Knowledge; Lasers; Leber' s Hereditary Optic Neuropathy; Life; Link; MELAS Syndrome; Malignant Neoplasms; Mammalian Cell; Mammalian Genetics; Measures; Medicine; Membrane; Metabolic; Metabolism; Metals; Microfluidics; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Molecular Biology; Muscle; Mutation; Names; Neurodegenerative Disorders; Nuclear; Nucleic Acids; Organ; Organelles; Parkinson Disease; Pathologic; Pharmaceutical Preparations; Phenotype; Physiologic pulse; Point Mutation; Positioning Attribute; Process; Production; Proteins; Public Health; Respiration; Ribosomal RNA; Shapes; Signal Transduction; Source; Speed; Stochastic Processes; Symptoms; Syndrome; System; Therapeutic; Thick; Thinness; Time; Tissues; Touch sensation; Transfer RNA; Translations; cell type; comparative; dietary supplements; effective therapy; experimental study; gene therapy; human disease; metallicity; mitochondrial DNA mutation; mitochondrial dysfunction; mitochondrial genome; oxidative damage; protein aggregate; public health relevance; repaired; reverse genetics; skills; small molecule; success; theories

 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.

 描述（由申请人提供）：线粒体是哺乳动物细胞的基本细胞器。它们为生物合成过程产生能量（ATP）和TCA循环代谢物，调节细胞内Ca 2+通量和Fe-S簇合成，并启动细胞凋亡。为了组装一个线粒体，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，超过1,000种蛋白质中只有13种是由母系遗传的~16.6kb mtDNA编码的，但这13种蛋白质是细胞呼吸的电子传递链的重要组成部分。影响这13种蛋白质的翻译、组装或功能的mtDNA突变会导致超过200种已命名的线粒体疾病综合征，这些综合征会影响高能量器官，如大脑、肌肉或心脏，并经常导致过早死亡。不幸的是，没有有效的治疗方法或支持措施用于mtDNA疾病。主要的希望是最终纠正或补偿有害的mtDNA突变。然而，与相对容易获得的改变gDNA序列的途径相比，改变mtDNA存在几乎完全的场阻断。 许多实验室正在试图开发线粒体反向遗传学，其中改变mtDNA产生研究的表型。然而，目前的方法是低效的，控制不良的随机过程，往往与伦理问题的细胞源材料。几个实验室已经成功地分离、修饰并在体外将改变的mtDNA重新引入线粒体中，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工程改造的线粒体重新引入细胞中进行功能性、系统性研究。 在这里，我们建议启用线粒体反向遗传学，并提供一个初步的方法来纠正破坏性的mtDNA突变。在长期的合作中，Chiou和Teitell实验室发明了一种光热纳米叶片，可以将天然或工程线粒体转移到哺乳动物细胞中，并挽救细胞呼吸的缺陷。然而，我们目前的纳米刀片所需的技能，缓慢的速度和庞大的系统尺寸导致许多失败的实验，并排除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3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，微流体平台，用于大规模并行线粒体递送，我们称之为BLAST。在目标2中，我们将部署BLAST来产生或纠正特定的mtDNA突变，这些突变导致3种具有天然线粒体转移的人类疾病综合征。在目标3中，我们将改变线粒体DNA，并利用BLAST将体外修饰的线粒体转移回细胞中以产生杂交细胞系，以彻底评估功能活性，包括不可能进行的全系统碳示踪研究。结合起来，我们的工程和分子生物学跨学科方法将使线粒体DNA的靶向改变既为基础，基础研究和线粒体医学的未来翻译应用的开始。