Chapter 19 - Mitochondrially targeted zinc finger nucleases

Abstract

Mutations in protein or RNA coding genes of human mitochondrial DNA (mtDNA) are the most frequent cause of mitochondrial disease. These disorders are currently incurable and effectively untreatable, with heterogeneous penetrance, presentation, and prognosis. In most cases, mutant- and wild-type mtDNAs coexist within a single cell, resulting in heteroplasmy. The selective elimination of mutant mtDNA, and consequent enrichment of wild-type mtDNA, can rescue pathological phenotypes in heteroplasmic cells. In this chapter, we describe the development and application of mitochondrially targeted zinc finger nucleases (mtZFNs) for degradation of mutant mtDNA through site-specific DNA cleavage. We have successfully used mtZFNs to target and cleave mtDNA harboring disease-associated point mutations or large-scale deletions in vitro. More recently, we have used a mouse model that recapitulates common molecular features of heteroplasmic mtDNA disease to target mtDNA mutations in vivo. We delivered mtZFNs systemically by adeno-associated virus (AAV), inducing specific reduction of mutant mtDNA and improvement in molecular and biochemical phenotypes. These data constitute proof of concept that mtDNA heteroplasmy correction using mtZFNs could provide a therapeutic route for heteroplasmic mitochondrial diseases.