Optimization of mtDNA-targeted platinum TALENs for bi-directionally modifying heteroplasmy levels in patient-derived m.3243A>G-iPSCs

Patient-derived induced pluripotent stem cells (iPSCs) are a useful pathological model for debilitating diseases caused by mitochondrial DNA (mtDNA) mutations. We established iPSCs derived from mitochondrial disease patients, heteroplasmic for the m.3243A>G mutation. The proportion of a selected mtDNA can be reduced by delivering a programmable nuclease into the mitochondria, and we developed various mtDNA-targeted Platinum TALENs (mpTALENs) to modify m.3243A>G-iPSC heteroplasmy levels in either wild-type or mutant direction. For TALEN optimization, the use of non-conventional repeat-variable di-residues (ncRVD)—LK/WK or NM—enhanced cleavage activity and specificity, and the replacement of conventional with obligate heterodimeric FokI nuclease domains increased target specificity and protected mtDNA from copy number depletion. In vitro, depending on whether wild-type or mutant mtDNA was targeted, we could obtain m.3243A>G-iPSCs with a higher or lower mutation load, while the cells retained their ability to differentiate into three germ layers. These results demonstrate that our mpTALEN optimization created a useful tool for altering heteroplasmy levels in m.3243A>G-iPSCs, improving the potential for studying mutation pathology. The enhanced efficiency also holds promise for using m.3243G(MUT)-mpTALEN as a therapeutic strategy for treating patients suffering from m.3243A>G mitochondrial diseases.