Building the mitochondrial genome editing repertoire

构建线粒体基因组编辑库

• 批准号: 10220697
• 负责人: KARL J CLARK
• 金额: $ 39.75万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-21 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10220697
• 关键词: Address; Aging; Apoptosis; Binding; Biochemical Process; Cell Nucleus; Cells; DNA; DNA Binding; DNA Sequence Alteration; DNA cassette; Deoxyribonucleases; Development; Disease; Encephalopathies; Environment; Enzymes; Event; Family; Genes; Genome; Genome engineering; Genotype; Goals; Homeostasis; Human; Human Genome; Kearns-Sayre syndrome; Lactic Acidosis; Measures; Metabolism; Methods; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial Encephalomyopathies; Mitochondrial Myopathies; Mitochondrial RNA; Molecular; Mutation; Myopathy; Nuclear; Nucleic Acids; Organ; Output; Pathogenicity; Pathway interactions; Patients; Physiology; Plants; Play; Point Mutation; Protein Import; Proteins; RNA; RNA Binding; RNA Editing; RNA Recognition Motif; Reagent; Reporter; Ribonucleases; Role; Somatic Cell; Stroke; Syndrome; System; Tertiary Protein Structure; Testing; Therapeutic; Transcript; Tube; Variant; Work; base; genome editing; heteroplasmy; mitochondrial genome; novel; programs; repaired; response; scaffold; somatic cell gene editing; therapeutic genome editing; tool

Abstract Mitochondria have critical normal roles in metabolism, organ homeostasis, apoptosis and aging. Mitochondria play important but still largely mysterious roles in human physiology. Mutations in both nuclear DNA associated with proteins imported into mitochondria as well as mitochondrial DNA (mtDNA) are pathogenic. Despite this clear association of genotype with disease, there are no current treatments for patients with mitochondrial disease. Mitochondria represent a unique cellular compartment with different DNA and RNA repair and editing rules. For example, DNA nucleases that introduce double strand breaks and subsequent repair in nuclear DNA induce the degradation of mtDNA. Indeed, none of the common repair pathways found in the nucleus are active in mitochondria. This proposal uses the well-established TALE-based programmable DNA binding system for targeting mtDNA and the single-tube FUSX TALE assembly system to rapidly generate any protein-based genome engineering reagents. Similarly, we use a new, protein-based and programmable RNA binding system based on PPR proteins, a class of naturally occurring, mitochondrially localized RNA editors from plants. This application harnesses the unique environment of mitochondria to generate a new toolbox to expand the repertoire of tools to edit the human genome (RFA-RM-18-017). To develop these new molecular reagents for mtDNA and mtRNA editing of somatic cells, we will conduct the following aims: I. Develop new classes of mtDNA editing tools. Enhanced approaches to the use of mitoTALENs for preferential degradation of pathogenic mtDNA variants for MELAS and KSS will be developed, including novel nuclearly encoded reporters to detect non-mitochondrial off-targeting gene editing events. A new class of TALE mitochondrial base editors will be developed to directly edit mtDNA for pathogenic variants. II. mtRNA editing tools will be generated through harnessing the PPR family of naturally occurring programmable RNA editors. We will use our new FUSR assembly system to rapidly develop optimal RNA binding reagents, including the fusion to a set of test RNA nuclease or editing protein domains. Errant fusion transcripts in mtDNA deletion or single base variants in heteroplasmic cells will be used as the test paradigm for potential RNA editing platform development with the potential use as a therapeutic. Milestones for initial stages include the establishment of novel mtDNA heteroplasmy converting mitoTALENs against MELAS and KSS followed by testing of the new mtDNA base editor. For mtRNA editing, establishing PPR scaffolding rules for mtRNA binding followed by new programmable RNA nucleases and editors will be established. Deliverables include these novel mtDNA and mtRNA editing systems as well as humans cells with matched nuclearly encoded off-target reporter cassettes for use by any mitochondrial gene editing therapeutic system.

抽象的 线粒体在代谢，器官稳态，凋亡和衰老中具有关键的正常作用。线粒体 在人类生理学中发挥重要但在很大程度上仍然是神秘的作用。两个核DNA的突变 将进口到线粒体以及线粒体DNA（mtDNA）的蛋白质是致病性的。尽管如此 基因型与疾病的明确关联，目前尚无针对线粒体患者的治疗 疾病。 线粒体代表一个独特的细胞室，具有不同的DNA和RNA修复和编辑 规则。例如，引入双链断裂和随后在核DNA进行修复的DNA核酸酶 诱导mtDNA的降解。确实，核中没有发现的常见修复途径是 活跃在线粒体中。 该提案使用基于故事的可编程DNA结合系统来定位 mtDNA和单管FUSX故事组装系统，以快速生成任何基于蛋白质的基因组 工程试剂。同样，我们使用基于蛋白质的新的，可编程的RNA结合系统 在PPR蛋白上，一类天然存在的线粒体定位的RNA编辑器来自植物。 该应用程序利用线粒体的独特环境生成一个新的工具箱 扩展编辑人类基因组的工具曲目（RFA-RM-18-017）。开发这些新的 用于MTDNA和MTRNA的分子试剂，分子细胞的MTRNA编辑，我们将执行以下目的： I.开发新的MTDNA编辑工具。增强了使用Mitotalens的方法 将开发用于MELA和KSS的致病性mtDNA变体的优先降解，包括 新型的核编码记者检测非靶向基因编辑事件的非靶向基因。一个新 将开发一类故事线粒体基础编辑器，以直接为致病性变体编辑mtDNA。 ii。 MTRNA编辑工具将通过利用自然发生的PPR家族而生成 可编程RNA编辑器。我们将使用新的FUSR组装系统快速发展最佳RNA 结合试剂，包括与一组测试RNA核酸酶或编辑蛋白结构域的融合。错误 MTDNA缺失或杂质细胞中单基碱变异的融合转录本将用作测试 潜在的RNA编辑平台开发的范例，并具有潜在用作治疗性。 初始阶段的里程碑包括建立新型mtDNA杂质转换有丝分析 反对Melas和KSS，然后测试新的MTDNA基本编辑器。用于MTRNA编辑，建立 MTRNA结合的PPR脚手架规则，然后是新的可编程RNA核酸酶，编辑将是 已确立的。可交付成果包括这些新颖的mtDNA和mTRNA编辑系统以及人类细胞 与匹配的核编码脱靶记者盒一起用于任何线粒体基因编辑 治疗系统。