Application Note

Mitochondria, the so-called “powerhouses” of cells, are double-membrane cellular organelles that are found in almost all eukaryotic cells. Mutations in mtDNA occur at a higher rate than in nuclear DNA, and mutations in mtDNA cause a group of maternally inherited genetic disorders termed mitochondrial diseases, which affect 1 in 5,000 live births and cause significant morbidity and mortality.

In most mitochondrial diseases, mutant mtDNA co-exists with wild-type mtDNA, resulting in a situation of mtDNA heteroplasmy in which the residual wild-type mtDNA can partially compensate for the mutated mtDNA, averting a complete bioenergetic crisis. However, when the percentage of mutant mtDNA exceeds a threshold in the range of 60%–95%, depending on the severity of the mutation, pathogenic mtDNA mutations can lead to a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases may provide an alternative avenue for treating mitochondrial disorders via targeted destruction of the mutant mtDNA and induction of heteroplasmic shifting.

Here, researchers at Guangzhou Medical University generated mitochondrial disease patient-specific induced pluripotent stem cells (MiPSCs) that harbored a high proportion of m.3243A>G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes (MELAS). They engineered mitochondrial-targeted transcription activator-like effector nucleases (mitoTALENs) to specificly target mutant mtDNA and tested the mitoTALENs in MiPSC5 sub-lines harboring approximately 90% mutant m.3243A>G mtDNA.

To quantify the heteroplasmic shifting effect (3243A>G mutation ratio) of the the mitoTALENs on the MiPSC5 and the sub-clones, the researchers performed targeted sequencing of the entire mtDNA genome using the VariantPro Mitochondrial Panel A panel designed for 100% amplicon coverage (16,569 bp) of the mitochondrion genome.

They found 92% and 89% 3243A>G heteroplasmy was detected in the MiPSC5 and TALEN-control cells, respectively, 27% 3243A>G heteroplasmy was detected in the targeted MiPSC5-T1 sub-clone, and the targeted MiPSC5-T3 and MiPSC5-T7 cells were homoplasmic for the wild-type allele.

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(Left) Percentages of A and G reads at the mtDNA 3,243 position of the MELAS-iPSCs and targeted subclones were quantified using next-generation sequence analysis. (Right) Single-nucleotide variations (SNVs) in iPSCs via VariantPro sequencing. The black bars represent individual SNVs. Compared with untargeted b-thal iPSCs, iPSC-C2 had 12 SNVs, and the remaining corrected colonies had 21 SNVs.

In addition to the pathogenic 3243A>G mutations, two heteroplasmic variants in the 16S rRNA gene and in MT-ATP6 were different in the MiPSC5 and targeted sub-clones. In addition, in all of those clones, they also detected another 7 single nucleotide polymorphisms (SNPs) in the D-loop region, 2 in the 12S rRNA gene, 2 in the 16S rRNA gene, 1 in the tRNA-R gene, and 23 in protein genes. Clinical symptoms associated with these variants have not been reported.

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This study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA in iPSCs, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA.

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