Pathogenic mitochondrial DNA (mtDNA) mutations often co‐exist with wild‐type molecules (mtDNA heteroplasmy). Phenotypes manifest when the percentage of mutant mtDNA is high (70–90%). Previously, our laboratory showed that mitochondria‐targeted transcription activator‐like effector nucleases (mitoTALENs) can eliminate mutant mtDNA from heteroplasmic cells. However, mitoTALENs are dimeric and relatively large, making it difficult to package their coding genes into viral vectors, limiting their clinical application. The smaller monomeric GIY‐YIG homing nuclease from T4 phage (I‐TevI) provides a potential alternative. We tested whether molecular hybrids (mitoTev‐TALEs) could specifically bind and cleave mtDNA of patient‐derived cybrids harboring different levels of the m.8344A>G mtDNA point mutation, associated with myoclonic epilepsy with ragged‐red fibers (MERRF). We tested two mitoTev‐TALE designs, one of which robustly shifted the mtDNA ratio toward the wild type. When this mitoTev‐TALE was tested in a clone with high levels of the MERRF mutation (91% mutant), the shift in heteroplasmy resulted in an improvement of oxidative phosphorylation function. mitoTev‐TALE provides an effective architecture for mtDNA editing that could facilitate therapeutic delivery of mtDNA editing enzymes to affected tissues.

Diseases caused by heteroplasmic mitochondrial DNA mutations have no effective treatment or cure. In recent years, DNA editing enzymes were tested as tools to eliminate mutant mtDNA in heteroplasmic cells and tissues. Mitochondrial-targeted restriction endonucleases, ZFNs, and TALENs have been successful in shifting mtDNA heteroplasmy, but they all have drawbacks as gene therapy reagents, including: large size, heterodimeric nature, inability to distinguish single base changes, or low flexibility and effectiveness. Here we report the adaptation of a gene editing platform based on the I-CreI meganuclease known as ARCUS®. These mitochondrial-targeted meganucleases (mitoARCUS) have a relatively small size, are monomeric, and can recognize sequences differing by as little as one base pair. We show the development of a mitoARCUS specific for the mouse m.5024C>T mutation in the mt-tRNAAla gene and its delivery to mice intravenously using AAV9 as a vector. Liver and skeletal muscle show robust elimination of mutant mtDNA with concomitant restoration of mt-tRNAAla levels. We conclude that mitoARCUS is a potential powerful tool for the elimination of mutant mtDNA.

Establishing the molecular basis of DNA mutations that cause inherited disease is of fundamental importance to understanding the origin, nature, and clinical sequelae of genetic disorders in humans. The majority of disease-associated mutations constitute single-base substitutions and short deletions and/or insertions resulting from DNA replication errors and the repair of damaged bases. However, pathological mutations can also be introduced by nonreciprocal recombination events between paralogous sequences, a phenomenon known as interlocus gene conversion (IGC). IGC events have thus far been linked to pathology in more than 20 human genes. However, the large number of duplicated gene sequences in the human genome implies that many more disease-associated mutations could originate via IGC. Here, we have used a genomewide computational approach to identify disease-associated mutations derived from IGC events. Our approach revealed hundreds of known pathological mutations that could have been caused by IGC. Further, we identified several dozen highconfidence cases of inherited disease mutations resulting from IGC in~1% of all genes analyzed. About half of the donor sequences associated with such mutations are functional paralogous genes, suggesting that epistatic interactions or differential expression patterns will determine the impact upon fitness of specific substitutions between duplicated genes. In addition, we identified thousands of hitherto undescribed and potentially deleterious mutations that could arise via IGC. Our findings reveal the extent of the impact of interlocus gene conversion upon the spectrum of human inherited disease.

Mutations in the mitochondrial genome are the cause of many debilitating neuromuscular disorders. Currently, there is no cure or treatment for these diseases, and symptom management is the only relief doctors can provide. Although supplements and vitamins are commonly used in treatment, they provide little benefit to the patient and are only palliative. This is why gene therapy is a promising research topic to potentially treat and, in theory, even cure diseases caused by mutations in the mitochondrial DNA (mtDNA). Mammalian cells contain approximately a thousand copies of mtDNA, which can lead to a phenomenon called heteroplasmy, where both wild‐type and mutant mtDNA molecules co‐exist within the cell. Disease only manifests once the per cent of mutant mtDNA reaches a high threshold (usually >80%), which causes mitochondrial dysfunction and reduced ATP production. This is a useful feature to take advantage of for gene therapy applications, as not every mutant copy of mtDNA needs to be eliminated, but only enough to shift the heteroplasmic ratio below the disease threshold. Several DNA‐editing enzymes have been used to shift heteroplasmy in cell culture and mice. This review provides an overview of these enzymes and discusses roadblocks of applying these to gene therapy in humans.

No abstract