Targeted elimination of mutant mitochondrial DNA in MELAS-iPSCs by mitoTALENs

Abstract

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.

Can Mitochondrial DNA be CRISPRized: Pro and Contra

“…Last years, several alternative approaches to manipulate mtDNA were developed. One successful strategy was based on the TALEN technology, adapted for mitochondria (mito‐TALENs) , the second – on sequence‐specific designed Zn‐fingers nucleases targeted in the organelles . As one of the largely used approach to manipulate DNA in vivo is currently the CRISPR system (in all its versions), the legitimate idea was to apply it to mitochondria (discussed in Reference ).…”

Mitochondrial DNA heteroplasmy in disease and targeted nuclease‐based therapeutic approaches

“…Further, both our group and the Minczuk group were able to use mitoTALEN and mitoZFN to effectively shift mtDNA heteroplasmy in cell models of the common deletion . mitoTALENs have also been used in two different iPSC models of mutations associated with MELAS, where transfection of mitoTALENs resulted in the reduction in the specific mutant haplotype .…”

Strategies for fighting mitochondrial diseases

“…dimeric endonucleases such as Fok I) to selectively destroy mutated mtDNA, preserving wild‐type molecules. Both mitochondrial targeted (mt)TALENs and mtZFNs were shown to be able to reduce the heteroplasmic load in several cellular models with mutations in mtDNA [113‐115], and in vivo in the same mouse models carrying a heteroplasmic mutation in the MT‐tRNA‐Ala gene [116,117].…”