title: Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs.
abstract: Mitochondrial diseases are frequently associated with mutations in mitochondrial DNA (mtDNA). In most cases, mutant and wild-type mtDNAs coexist, resulting in heteroplasmy. The selective elimination of mutant mtDNA, and consequent enrichment of wild-type mtDNA, can rescue pathological phenotypes in heteroplasmic cells. Use of the mitochondrially targeted zinc finger-nuclease (mtZFN) results in degradation of mutant mtDNA through site-specific DNA cleavage. Here, we describe a substantial enhancement of our previous mtZFN-based approaches to targeting mtDNA, allowing near-complete directional shifts of mtDNA heteroplasmy, either by iterative treatment or through finely controlled expression of mtZFN, which limits off-target catalysis and undesired mtDNA copy number depletion. To demonstrate the utility of this improved approach, we generated an isogenic distribution of heteroplasmic cells with variable mtDNA mutant level from the same parental source without clonal selection. Analysis of these populations demonstrated an altered metabolic signature in cells harbouring decreased levels of mutant m.8993T>G mtDNA, associated with neuropathy, ataxia, and retinitis pigmentosa (NARP). We conclude that mtZFN-based approaches offer means for mtDNA heteroplasmy manipulation in basic research, and may provide a strategy for therapeutic intervention in selected mitochondrial diseases.
authors.affiliation: MRC Mitochondrial Biology Unit, Cambridge, UK pag44@mrc-mbu.cam.ac.uk.
authors.forename: Payam A
authors.initials: PA
authors.lastname: Gammage
authors.affiliation: MRC Cancer Unit, Cambridge, UK.
authors.forename: Edoardo
authors.initials: E
authors.lastname: Gaude
authors.affiliation: MRC Mitochondrial Biology Unit, Cambridge, UK.
authors.forename: Lindsey
authors.initials: L
authors.lastname: Van Haute
authors.affiliation: MRC Mitochondrial Biology Unit, Cambridge, UK GABBA, University of Porto, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Rebelo-Guiomar
authors.affiliation: MRC Mitochondrial Biology Unit, Cambridge, UK.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Jackson
authors.affiliation: MRC Mitochondrial Biology Unit, Cambridge, UK.
authors.forename: Joanna
authors.initials: J
authors.lastname: Rorbach
authors.affiliation: JDRF/Wellcome Trust DIL, Cambridge Institute for Medical Research, University of Cambridge, UK.
authors.forename: Marcin L
authors.initials: ML
authors.lastname: Pekalski
authors.affiliation: MRC Mitochondrial Biology Unit, Cambridge, UK.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Robinson
authors.affiliation: INSERM U1154, CNRS UMR 7196, Muséum National d'Histoire Naturelle, Paris, France.
authors.forename: Marine
authors.initials: M
authors.lastname: Charpentier
authors.affiliation: INSERM U1154, CNRS UMR 7196, Muséum National d'Histoire Naturelle, Paris, France.
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Concordet
authors.affiliation: MRC Cancer Unit, Cambridge, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Frezza
authors.affiliation: MRC Mitochondrial Biology Unit, Cambridge, UK mam@mrc-mbu.cam.ac.uk.
authors.forename: Michal
authors.initials: M
authors.lastname: Minczuk
mesh_terms: D045744:Cell Line, Tumor; D004272:DNA, Mitochondrial / Q000235:genetics*; D004720:Endonucleases / Q000378:metabolism*; D005434:Flow Cytometry; D018628:Gene Dosage; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D016337:RNA, Catalytic / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D004272:DNA, Mitochondrial; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D004720:Endonucleases