title: MitoTALEN: A General Approach to Reduce Mutant mtDNA Loads and Restore Oxidative Phosphorylation Function in Mitochondrial Diseases.
abstract: We have designed mitochondrially targeted transcription activator-like effector nucleases or mitoTALENs to cleave specific sequences in the mitochondrial DNA (mtDNA) with the goal of eliminating mtDNA carrying pathogenic point mutations. To test the generality of the approach, we designed mitoTALENs to target two relatively common pathogenic mtDNA point mutations associated with mitochondrial diseases: the m.8344A>G tRNA(Lys) gene mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) and the m.13513G>A ND5 mutation associated with MELAS/Leigh syndrome. Transmitochondrial cybrid cells harbouring the respective heteroplasmic mtDNA mutations were transfected with the respective mitoTALEN and analyzed after different time periods. MitoTALENs efficiently reduced the levels of the targeted pathogenic mtDNAs in the respective cell lines. Functional assays showed that cells with heteroplasmic mutant mtDNA were able to recover respiratory capacity and oxidative phosphorylation enzymes activity after transfection with the mitoTALEN. To improve the design in the context of the low complexity of mtDNA, we designed shorter versions of the mitoTALEN specific for the MERRF m.8344A>G mutation. These shorter mitoTALENs also eliminated the mutant mtDNA. These reductions in size will improve our ability to package these large sequences into viral vectors, bringing the use of these genetic tools closer to clinical trials.
authors.affiliation: Department of Neurology, University of Miami Miller School of Medicine Miami, Miami, Florida, USA.
authors.forename: Masami
authors.initials: M
authors.lastname: Hashimoto
authors.affiliation: Department of Neurology, University of Miami Miller School of Medicine Miami, Miami, Florida, USA.
authors.forename: Sandra R
authors.initials: SR
authors.lastname: Bacman
authors.affiliation: Department of Neurology, University of Miami Miller School of Medicine Miami, Miami, Florida, USA.
authors.forename: Susana
authors.initials: S
authors.lastname: Peralta
authors.affiliation: Division of Human Genetics, Department of Pediatrics, The Children's Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Marni J
authors.initials: MJ
authors.lastname: Falk
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.
authors.forename: Anne
authors.initials: A
authors.lastname: Chomyn
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Chan
authors.affiliation: Department of Neurology, University of Miami Miller School of Medicine Miami, Miami, Florida, USA.|Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, Florida, USA.
authors.forename: Sion L
authors.initials: SL
authors.lastname: Williams
authors.affiliation: Department of Neurology, University of Miami Miller School of Medicine Miami, Miami, Florida, USA.|Department of Cell Biology, University of Miami Miller School of Medicine, Miami, Florida, USA.
authors.forename: Carlos T
authors.initials: CT
authors.lastname: Moraes
mesh_terms: D000818:Animals; D002460:Cell Line; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D042967:Electron Transport Complex I / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage; D015870:Gene Expression; D023061:Gene Order; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D028361:Mitochondrial Diseases / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010085:Oxidative Phosphorylation; D017354:Point Mutation; D021381:Protein Transport; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; D014157:Transcription Factors; C481838:MT-ND5 protein, human; D003851:Deoxyribonucleases; D042967:Electron Transport Complex I