MitoTALEN: A General Approach to Reduce Mutant mtDNA Loads and Restore Oxidative Phosphorylation Function in Mitochondrial Diseases

(Objective) We have designed mitochondria-targeted Transcription Activator-Like Effector Nucleases or mitoTALENs to cleave specific sequences in the mitochondrial DNA (mtDNA) to eliminate mtDNA carrying pathogenic point mutations. (Method) We engineered mitoTALENs to target two relatively common pathogenic mtDNA point mutations associated with mitochondrial diseases: the m.8344A>G tRNALys gene mutation associated with Myoclonic Epilepsy with Ragged-Red Fibers (MERRF) and the m.13513G>A ND5 mutation associated with MELAS/Leigh Syndrome. Transmitochondrial cybrid cells harboring the respective heteroplasmic mtDNA mutations were transfected with the specific mitoTALEN and analyzed after different time periods. (Result) MitoTALENs efficiently reduced the levels of the targeted pathogenic mtDNAs in the respective cell lines. Functional assays showed that cells with heteroplasmic mutant mtDNA were able to recover respiratory capacity and oxidative phosphorylation enzymes activity after transfection with the mitoTALEN. We further designed shorter versions of the mitoTALEN specific for the MERRF m.8344A>G mutation. These shorter mitoTALENs also eliminated the mutant mtDNA. (Conclusion) These results show potential clinical applications of mitoTALEN for mitochondrial diseases.