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Keywords:

  • gene therapy;
  • heteroplasmy;
  • mitochondrial disease;
  • zinc finger nuclease

Abstract

We designed and engineered mitochondrially targeted obligate heterodimeric zinc finger nucleases (mtZFNs) for site-specific elimination of pathogenic human mitochondrial DNA (mtDNA). We used mtZFNs to target and cleave mtDNA harbouring the m.8993T>G point mutation associated with neuropathy, ataxia, retinitis pigmentosa (NARP) and the “common deletion” (CD), a 4977-bp repeat-flanked deletion associated with adult-onset chronic progressive external ophthalmoplegia and, less frequently, Kearns-Sayre and Pearson's marrow pancreas syndromes. Expression of mtZFNs led to a reduction in mutant mtDNA haplotype load, and subsequent repopulation of wild-type mtDNA restored mitochondrial respiratory function in a CD cybrid cell model. This study constitutes proof-of-principle that, through heteroplasmy manipulation, delivery of site-specific nuclease activity to mitochondria can alleviate a severe biochemical phenotype in primary mitochondrial disease arising from deleted mtDNA species.

Synopsis

Thumbnail image of graphical abstract

Mutations and rearrangements of mitochondrial DNA (mtDNA) are a common cause of human disease, where they often co-exist with wild-type mtDNA within a single cell. Mitochondrially targeted engineered zinc finger nucleases (mtZFNs) can phenotypically rescue a severe mtDNA-mediated dysfunction and show future therapeutic potential.

  • Previously reported mtZFN constructs were redesigned, greatly improving target specificity and allowing their safe use in human mitochondria.
  • The capacity of the novel mtZFN design was validated by selectively degrading point mutant mtDNA associated with neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) and maternally inherited Leigh syndrome (MILS).
  • The use of the novel mtZFNs was expanded by selectively degrading mtDNA harbouring a pathogenic large-scale deletion associated with adult-onset chronic progressive external ophthalmoplegia (CPEO) and, less frequently, Kearns-Sayre syndrome (KSS) and Pearson's marrow pancreas syndrome.
  • Data are provided demonstrating that elimination of deleted, pathogenic mtDNA molecules by mtZFNs is sufficient for full recovery of oxidative phosphorylation in a disease model cell line.