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Mitochondrial diseases are maternally inherited heterogeneous 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 destruction 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. Furthermore, 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.Electronic supplementary materialThe online version of this article (10.1007/s13238-017-0499-y) contains supplementary material, which is available to authorized users.

Electromechanical delay (EMD) was described as a time elapsed between first trigger and force output. Various results have been reported based on the measurement method with observed inconsistent results when the trigger is elicited by voluntary contraction. However, mechanomyographic (MMG) sensor placed far away on the skin from the contracting muscle was used to detect muscle fiber motion and excitation-contraction (EC) coupling which may give unreliable results. On this basis, the purpose of this study was to detect EMD during active muscle contraction whilst introducing an ultrafast ultrasound (US) method to detect muscle fiber motion from a certain depth of the muscle. Time delays between onsets of EMG-MMG, EMG-US, MMG-FORCE, US-FORCE, and EMG-FORCE were calculated as 20.5 ± 4.73, 28.63 ± 6.31, 19.21 ± 6.79, 30.52 ± 8.85, and 49.73 ± 6.99 ms, respectively. Intrarater correlation coefficient (ICC) was higher than MMG when ultrafast US was used for detecton of the Δt EMG-US and Δt US-FORCE, ICC values of 0.75 and 0.70, respectively. Synchronization of the ultrafast ultrasound with EMG and FORCE sensors can reveal reliable and clinically useful results related to the EMD and its components when muscle is voluntarily contracted. With ultrafast US, we detect onset from the certain depth of the muscle excluding the tissues above the muscle acting as a low-pass filter which can lead to inaccurate time detection about the onset of the contracting muscle fibers. With this non-invasive technique, understanding of the muscle dynamics can be facilitated.

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