Mitochondria are double membrane bound organelles that are found in all eukaryotic cells and carry a specialized circular 16.5kb genome. There is a group of devastating human genetic diseases caused by inherited or spontaneous mutation of genes encoded within the mitochondrial genome. These diseases can cause a wide array of medical problems from subtle muscular weakness to extreme neuromuscular problems such as loss of balance and coordination, seizures, stroke, dementia and death. Further, it is estimated that 1 in 10,000 children are diagnosed with a mitochondrial disease each year. Despite the importance of the mitochondrial genome in maintaining normal cellular function, as well as its role in promoting mitochondrial dysfunction when mutated, no method exists to alter the genome of mitochondria in a site-specific manner, until now. We have recently developed the first reliable method to introduce specific mutations into the mitochondrial genome of vertebrate animal cells. We have found that two pairs of site specific DNA-binding transcription activator-like (TAL) domain endo-nickases (mitoTALE-nicakses), can be directed to the mitochondria and that their activity results in generation of an intact mitochondrial genome, but with a deletion between the sites of the mtDNA nicks. Using a second pair of mitochondrial-targeted TAL endonucleases (mitoTALENs) to make a double strand DNA break in between the nick sites, we can induce the selective loss of wild-type mitochondrial genomes which did not undergo the deletion. Thus, we can seed mtDNA deletions using mitoTALE-nickases and enrich for edited mtDNA genomes using mitoTALENs. We have efficiently induced mtDNA deletions in zebrafish embryos and human and mouse cell lines. In this proposal, we will extend the innovations in mammalian cells by engineering targeted and precise mtDNA deletions systemically in mice. We will also develop inducible systems for expressing the mitoTALENs, which means we will make it possible to create “conditional” loss of function of mitochondrial genes in mice. This inducible mouse model system could be highly useful in settings in which the mitochondrial deletion would cause a selective disadvantage to such cells during early mouse development. We will create mice carrying an inducible deletion that causes Kearns Sayre syndrome (KSS) in humans, one of the most common mitochondrial genetic disease. The production of these novel mouse models will be useful for academic and pharmaceutical companies to test novel regenerative stem cell technologies.
Public Health Relevance Statement: Project Narrative Although there have been significant advancements in gene editing over the past two decades, none have been able to precisely edit the mitochondrial genome, until now. The goal of the proposed project is to use mitochondrial gene editing technology to generate an inducible mouse model of mitochondrial genetic disease. This project will result in a set of mouse models necessary to identify and test novel therapeutics for the treatment of mitochondrial disease.
Project Terms: 3T3 Cells; Animal Model; Autistic Disorder; Autoimmunity; Biological Models; Biology; Cardiovascular Diseases; Cell Line; Cell physiology; Cells; cellular engineering; Cessation of life; Chemicals; Child; Clinical; commercial application; Cytoplasm; Data; Dementia; Development; Diagnosis; Disadvantaged; Disease; Disease model; disease-causing mutation; DNA; DNA Binding; DNA Double Strand Break; DNA Sequence Alteration; Doxycycline; Embryo; endonuclease; Endonuclease I; Equilibrium; Eukaryotic Cell; functional genomics; Gene Mutation; Generations; Genes; Genetic Diseases; genetic manipulation; Genetic Models; Genome; genomic tools; Goals; Human; Human Biology; human disease; Human Genetics; improved; induced pluripotent stem cell; Inherited; innovation; Kearns-Sayre syndrome; loss of function; Mammalian Cell; Medical; Membrane; Messenger RNA; Methods; Mitochondria; Mitochondrial Diseases; Mitochondrial DNA; mitochondrial DNA mutation; mitochondrial dysfunction; mitochondrial genome; Modeling; Mouse Cell Line; mouse development; mouse model; Mus; Muscle Weakness; mutant; Mutate; Mutation; Neoplasm Metastasis; neuromuscular; novel; novel therapeutics; Nuclear; Organelles; Patients; Pharmacologic Substance; Phase; phase 2 study; Phenotype; Play; Process; Production; regenerative; Reproducibility; Research; Role; Seeds; Seizures; Services; Severities; Site; Source; stem cell technology; Stroke; Symptoms; System; T-Lymphocyte; targeted nucleases; Technology; Testing; tool; transcription activator-like effector nucleases; Transcription Coactivator; Transgenic Mice; UV Mutagenesis; vector; Vertebrates; Zebrafish
