摘要
Mitochondrial diseases are maternally inherited hetero- geneous disorders that are primarily caused by mitochondrial DNA （mtDNA） mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruc- tion of the mutant mtDNA and induction of heteroplasmic shifting. Here, we 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）. We engineered mitochondrial-targeted transcription activator-like effector nucleases （mitoTALENs） and successfully eliminated the m.3243A〉G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Further- more, we successfully achieved reduction in the human m.3243A〉G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, 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.
Mitochondrial diseases are maternally inherited hetero- geneous disorders that are primarily caused by mitochondrial DNA （mtDNA） mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruc- tion of the mutant mtDNA and induction of heteroplasmic shifting. Here, we 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）. We engineered mitochondrial-targeted transcription activator-like effector nucleases （mitoTALENs） and successfully eliminated the m.3243A〉G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Further- more, we successfully achieved reduction in the human m.3243A〉G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, 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.
基金
This work was supported in part by the ＂Reproductive health and major birth defects prevention and control research＂ Key Special Fund （No. 2016YFC1000601）, the National Natural Science Foundation of China （Grant Nos. 31371521, 81370766, 81401254, 81570101, 81671121, 31601187, 81521002）, the Guangdong Province Science and Technology Project （2014TQ01R683, 2017A020 214005, 2016A020216023, 2015A030310119, 2016B030229008）, the Bureau of Science and Technology of Guangzhou Municipality （201505011111498）, the ＂Reproductive health and major birth defects prevention and control research＂ Key Special Fund （Nos. 2016YFC1000201 and 2016YFC1000302）, the Ministry of Science and Technology of China Grants （973 program
2014CB943203）, and the Beijing Nova Program （xxjh2015011）.
