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Building the mitochondrial genome editing repertoire

构建线粒体基因组编辑库

基本信息

批准号：
9767023
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
项目类别：
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/9767023
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 mitochondrial genome novel programs repaired response scaffold somatic cell gene editing tool

项目摘要

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