Many mitochondrial diseases have been difficult to study and treat due to the inherent challenges in accessing mitochondrial DNA (mtDNA). Now, researchers from Japan have optimized mitochondrial-targeted compounds that can selectively modify the ratio of normal versus mutant mtDNA in patient-derived stem cells. This technology enables the creation of research models with varying mutation loads and demonstrates potential as a therapeutic strategy for reducing mutant mtDNA in patients, offering hope for mitochondrial disease treatment.

Mitochondrial diseases affect approximately one in 5,000 people worldwide, causing debilitating symptoms ranging from muscle weakness to stroke-like episodes. Some of these conditions result from mutations in mitochondrial DNA (mtDNA), the genetic material housed in these organelles. For patients with the common m.3243A>G mutation, which can cause MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) and diabetes mellitus, treatments remain limited. A fundamental challenge in mitochondrial disease research is that patients typically have a mix of both normal and mutated mtDNA within their cells. This condition, known as heteroplasmy, makes targeted therapies difficult to develop, as the normal-to-mutated mtDNA ratios can vary greatly from tissue to tissue.

Additionally, current basic research into mtDNA mutations faces significant obstacles that stem from a lack of disease models. The complex relationship between mutation load (the per centage of mutated mtDNA) and disease severity remains poorly understood, in part because there are no tools to precisely manipulate heteroplasmy levels in either direction. Without the ability to create cellular models with different mutation loads, scientists cannot effectively study how varying per centages of mutated mtDNA relate to disease manifestation.

Against this backdrop, a research team led by Senior Assistant Professor Naoki Yahata from the Department of Developmental Biology, Fujita Health University School of Medicine, Japan, has developed a technology that can modify heteroplasmy levels in cultured cells carrying the m.3243A>G mutation. Their paper was made available online on March 20, 2025, and will be published in Volume 36, Issue 2 of the journal Molecular Therapy Nucleic Acids on June 10, 2025. It was co-authored by Dr. Yu-ichi Goto from the National Center of Neurology and Psychiatry and Dr. Ryuji Hata from Osaka Prefectural Hospital Organization. In it, they detail the engineering and use of optimized mtDNA-targeted platinum transcription activator-like effector nucleases (mpTALENs) – specialized enzymes that can selectively target and cleave specific DNA sequences.

The researchers first established cultures of patient-derived induced pluripotent stem cells (iPSCs) containing the m.3243A>G mutation and then designed two versions of their mpTALEN systems: one that targets mutant mtDNA for destruction and another that targets normal mtDNA. This bi-directional approach allowed them to generate cells with mutation loads ranging from as low as 11 per cent to as high as 97 per cent, while still maintaining the cells’ ability to differentiate into various tissue types. “Our study is the first to demonstrate an increase in the proportion of pathogenic mutant mtDNA by programmable nuclease,” notes
Dr. Yahata.

Key innovations in their approach included the use of novel non-conventional repeat-variable di-residues and obligate heterodimeric FokI nuclease domains, which enhanced the technology’s specificity and reduced unwanted degradation of off-target mtDNA. The team also employed additional techniques, such as uridine supplementation, to establish stable cell lines with different mutation loads, even those that might typically have a growth disadvantage. “Our results demonstrate that our mpTALEN optimization process created a useful tool for altering heteroplasmy levels in m.3243A>G-iPSCs, improving their potential for studying mutation pathology. This enhanced efficiency also holds promise for using mpTALENs in therapeutic strategies for treating patients suffering from m.3243A>G mitochondrial diseases,”
says Dr. Yahata.

Overall, the study represents a significant advancement in mitochondrial medicine for several reasons. First, it provides researchers with multiple isogenic – otherwise genetically identical – cell lines that differ only in their level of heteroplasmy. This allows for a precise study of how mutation load affects disease manifestation. Second, it suggests that mpTALEN technology may become therapeutically valuable for reducing mutant mtDNA load in patients.

“Our proposed method could be adapted for other mutant mtDNAs and may contribute to understanding their associated pathologies and developing new treatments, potentially benefiting patients with various forms of mitochondrial disease,” concludes Dr. Yahata.