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.

摘要 线粒体在代谢、器官稳态、细胞凋亡和衰老中具有关键的正常作用。线粒体 在人类生理学中扮演着重要但仍很神秘的角色。两种核DNA的突变 蛋白质输入线粒体以及线粒体DNA（mtDNA）是致病的。尽管如此 基因型与疾病的明确关联，目前还没有针对线粒体疾病患者的治疗方法。 疾病 线粒体代表了一个独特的细胞区室，具有不同的DNA和RNA修复和编辑 规则例如，在核DNA中引入双链断裂和随后修复的DNA核酸酶 诱导线粒体DNA降解。事实上，在细胞核中发现的常见修复途径没有一个是 活跃于线粒体中。 该提案使用完善的基于TALE的可编程DNA结合系统用于靶向 mtDNA和单管FUSX TALE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，我们使用一种新的，基于蛋白质和可编程的RNA结合系统， PPR蛋白是一类天然存在的、植物中的RNA编辑器。 该应用程序利用线粒体的独特环境来生成新的工具箱， 扩展编辑人类基因组的工具库（RFA-RM-18-017）。开发这些新的 为mtDNA和体细胞的mtDNA编辑的分子试剂，我们将进行以下目标： I.开发新的mtDNA编辑工具。使用mitoTALEN的改进方法 将开发针对MELAS和KSS的致病性mtDNA变体的优先降解，包括 新的核编码的报告基因，以检测非线粒体脱靶基因编辑事件。一个新 将开发一类TALE线粒体碱基编辑器以直接编辑mtDNA的致病性变体。 二. mtDNA编辑工具将通过利用PPR家族天然存在的 可编程RNA编辑器。我们将使用我们新的FUSR组装系统快速开发最佳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 将使用异质细胞中mtDNA缺失或单碱基变异的融合转录物作为试验 这是潜在的RNA编辑平台开发的范例，具有作为治疗剂的潜在用途。 初始阶段的重要性包括建立新的mtDNA异质性转化mitoTALEN 针对MELAS和KSS，然后测试新的mtDNA碱基编辑器。对于mtDNA编辑，建立 新的可编程RNA核酸酶和编辑器将遵循mtDNA结合的PPR支架规则， 确立了习这些新的线粒体DNA和线粒体RNA编辑系统以及人类细胞 具有匹配的核编码的脱靶报告盒，用于任何线粒体基因编辑 治疗系统