MitoTALENs: A general approach to reduce mutant mtDNA loads and restore oxidative phosphorylation function in mitochondrial diseases
We have designed mitochondrially targeted Transcription Activator-Like Effector Nucleases or mitoTALENs to cleave specific sequences in the mitochondrial DNA (mtDNA) with the goal of eliminating mtDNA carrying pathogenic point mutations. To test the generality of the approach we designed mitoTALENs to target two relatively common pathogenic mtDNA point mutations associated with mitochondrial diseases: the m.8344A>G tRNA^(Lys) 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 harbouring the respective heteroplasmic mtDNA mutations were transfected with the respective mitoTALEN and analysed after different time periods. 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. To improve the design in the context of the low complexity of mtDNA, we designed shorter versions of the mitoTALEN specific for the MERRF m.8344A>G mutation. These shorter mitoTALENs also eliminated the mutant mtDNA. These reductions in size improve our ability to package these large sequences into viral vectors, bringing the use of these genetic tools closer to clinical trials.
Additional Information© 2015 American Society of Gene & Cell Therapy. Under a Creative Commons license -- Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) Received 22 April 2015; Accepted 21 June 2015; Accepted article preview online 10 July 2015. We are grateful to the skilled assistance of the Flow Cytometry Core Facility for the cell sorting services and the Oncogenomics Core Facility for the Sanger sequencing services at the Sylvester Comprehensive Cancer Center, University of Miami. This work was supported by donations from Mr. Ron Biscardi, the JDM Fund and grants from the NIH (5R01EY010804 and 5R01NS079965), the Muscular Dystrophy Association and the United Mitochondrial Disease Foundation.
Supplemental Material - 1-s2.0-S1525001616302908-mmc1.pdf