Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs

Gammage, P. A. et al. (2016) Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs. Nucleic Acids Research, 44(16), pp. 7804-7816. (doi: 10.1093/nar/gkw676) (PMID:27466392) (PMCID:PMC5027515)

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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.

Item Type:Articles
Additional Information:Medical Research Council, UK; EMBO Fellowship [ALTF 701-2013 to L.V.H.]; PhD fellowship from the Foundation for Science and Technology, Portugal through the GABBA Program, University of Porto (to P.R.G.); Experiments undertaken in the J-PC laboratory were supported by ANR Investissement d'Avenir [ANR-IIINSB-0014] and AFM [18566]. Funding for open access charge: Medical Research Council, UK.
Glasgow Author(s) Enlighten ID:Gammage, Dr Payam
Authors: Gammage, P. A., Gaude, E., Van Haute, L., Rebelo-Guiomar, P., Jackson, C. B., Rorbach, J., Pekalski, M. L., Robinson, A. J., Charpentier, M., Concordet, J.-P., Frezza, C., and Minczuk, M.
College/School:College of Medical Veterinary and Life Sciences > School of Cancer Sciences
Journal Name:Nucleic Acids Research
Publisher:Oxford University Press
ISSN (Online):1362-4962
Published Online:27 July 2016
Copyright Holders:Copyright © 2016 The Authors
First Published:First published in Nucleic Acids Research 44(16):7804-7816
Publisher Policy:Reproduced under a Creative Commons License

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