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

Payam A Gammage,Jean-Paul Concordet,Edoardo Gaude,Alan J Robinson,Marcin L Pekalski,Michal Minczuk,Pedro Rebelo-Guiomar,Christopher B Jackson,Lindsey Van Haute,Christian Frezza,Marine Charpentier,Joanna Rorbach

doi:10.1093/nar/gkw676

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.

Highlights

Mitochondria are ubiquitous organelles within the eukaryotic domain, acting as a hub for numerous metabolic pathways and biochemical processes, most notably that of oxidative phosphorylation (OXPHOS)
We addressed the feasibility of targeting pathogenic multi-copy DNA genome (mtDNA) mutations using short-term, high expression of mitochondrially targeted zinc finger-nuclease (mtZFN)
A previously published mtZFN pairing shown to be specific to the m.8993T>G mutation, NARPd(+) and COMPa(−) [21,24], were cloned into vectors that co-express fluorescent marker proteins (mCherry for mtZFN(+), GFP for mtZFN(−)) enabling fluorescence activated cell sorting (FACS) of transiently transfected cells (Figure 1A and B)

Summary

INTRODUCTION

Mitochondria are ubiquitous organelles within the eukaryotic domain, acting as a hub for numerous metabolic pathways and biochemical processes, most notably that of oxidative phosphorylation (OXPHOS). Such degradation results in a heteroplasmic shift, altering the proportion of mutated : wild-type mtDNA following restoration of copy number The validity of this approach was first ascertained using mitochondrially targeted restriction endonucleases (mtREs), re-directed prokaryotic enzymes that bind and cleave specific DNA sequences with exceptional efficiency and fidelity [5,6,7,8,9,10,11,12]. Only a very limited number of disease-causative mutations produce unique restriction sites within mtDNA, and restriction endonucleases are essentially impossible to reengineer, either ab initio or in situ Another reagent, capable of such selective degradation, is the mitochondri-. We show that our improved mtZFN approach produces physiological rescue of the m.8993T>G model, revealing a substantial metabolic shift between healthy and disease states

MATERIALS AND METHODS

RESULTS

A FACS approach to modify dosage of mtZFN

DISCUSSION