Reverse Mitochondrial Genetics Enabled by Blast

Blast 实现反向线粒体遗传学

基本信息

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
项目类别：
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&apos 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&apos 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

Adoption Affect Aging Alpha Cell Alzheimer&apos 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&apos 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

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项目摘要

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（由适用提供）：线粒体是哺乳动物细胞的必不可少的细胞器。它们产生能量（ATP）和TCA循环代谢物进行生物合成过程，调节细胞内CA2+通量和Fe-S簇合成，并启动凋亡。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，构成线粒体的> 1,000个蛋白质中只有13个在主要遗传的〜16.6kb mtDNA中编码，但是这13种蛋白是电子传输链的必不可少的成分，可以实现细胞呼吸。影响这13种蛋白质的翻译，组装或功能的mtDNA突变导致> 200个称为线粒体疾病综合征，这些综合症会影响高能量器官，例如大脑，肌肉或心脏，并且常常导致早期死亡。不幸的是，没有有效的mtDNA疾病疗法或支持措施。主要希望是最终纠正或补偿有害的mtDNA突变。但是，与更改GDNA序列的相对访问相反，几乎完全可以改变mtDNA的磁场。许多实验室试图开发线粒体反向遗传学，其中改变mtdnas会产生用于研究的表型。但是，当前的方法效率低下，控制不良的随机过程通常对细胞源材料有道德问题。几个实验室已设法在体外隔离，修改和重新引入的mtDNA恢复到线粒体并显示转录活性，强烈建议将组装成核酸蛋白质聚集体中，称为核苷。但是，没有办法将这些MTDNA工程的线粒体重新引入细胞中进行功能，全系统研究。在这里，我们提出要实现线粒体反向遗传学，并为纠正毁灭性mtDNA突变提供了初始方法。在长期的合作中，Chiou和Teitell Labs发明了一种光热纳米刀片，可以将天然或工程的线粒体转移到哺乳动物细胞中，并在细胞呼吸中挽救缺陷。但是，我们目前的纳米刀片所需的技能，慢速和批量系统大小导致许多失败的实验，并且排除了这种方法的广泛采用。为了克服AIM 1，我们将生成一个高通量，紧凑的微流体平台，以大规模平行的线粒体传递，我们称为BLAST。在AIM 2中，我们将部署BLAST，以产生或纠正特定的mtDNA突变，这些突变引起3种具有天然线粒体转移的人类疾病综合征。在AIM 3中，我们将通过将体外修饰的线粒体转移回细胞中，以彻底评估功能活性，包括无法执行的碳跟踪研究，以彻底评估功能活性，从而改变mtDNA并利用爆炸来产生杂化细胞系。结合在一起，我们的工程和分子生物学跨学科方法将使MTDNA的靶向改变是基础，基础研究和线粒体医学中未来翻译应用的开始。