Scientists Engineer Precision Tool for Mitochondrial DNA Manipulation
A research team at Fujita Health University has developed a precision genome-editing approach to modulate levels of mutant mitochondrial DNA (mtDNA) in patient-derived stem cells, offering a new method to study mitochondrial diseases and potentially inform future therapeutic strategies. The findings, published in Molecular Therapy Nucleic Acids, center on engineered enzymes known as mpTALENs—mitochondria-targeted transcription activator-like effector nucleases.
Globally, mitochondrial diseases affect approximately 1 in 5,000 people, presenting with symptoms ranging from muscle weakness to stroke-like episodes. One of the major challenges in this field is the difficulty of manipulating the ratio of normal to mutant mtDNA (heteroplasmy), which hinders the development of precise disease models and therapies.
The researchers created two versions of mpTALENs: one designed to target and reduce mutant mtDNA, and another aimed at reducing normal mtDNA. This dual system allowed for bi-directional control over mtDNA mutation load, or heteroplasmy, within cells carrying the m.3243A>G mutation—a common cause of mitochondrial disorders such as MELAS syndrome and mitochondrial diabetes.
Using this method, the team generated isogenic induced pluripotent stem cell (iPSC) lines (used in biomedical research to isolate and study the effects of mutations) with mutation loads ranging from 11% to 97%, enabling more precise exploration of how different heteroplasmy levels influence disease mechanisms. Notably, the cells retained their ability to differentiate into multiple tissue types, which is essential for disease modeling.
The study is reported as the first demonstration of a programmable tool capable of increasing the proportion of pathogenic mtDNA, according to lead author Dr. Naoki Yahata, representing a departure from previous tools which have typically focused only on reducing mutant load.
Additional innovations included enhanced enzyme specificity, achieved through novel design elements that minimized off-target activity, and the use of uridine supplementation to support the growth of cells with higher mutation burdens. Specifically, uridine was added to compensate for metabolic deficits in high-mutation cells, helping maintain their viability despite a replication disadvantage.
The mpTALEN system employed non-conventional repeat-variable di-residues and obligate heterodimeric FokI nuclease domains—modifications that improved cleavage accuracy and reduced off-target degradation of mitochondrial DNA. The study emphasizes the value of this platform in generating reliable research models and highlights its potential relevance in developing future interventions to adjust heteroplasmy levels in patients.
While further research is needed to assess clinical applicability, the approach may also be adaptable to other mtDNA mutations, supporting broader efforts to better understand and eventually treat mitochondrial diseases.
The study (DOI: 10.1016/j.omtn.2025.102521) was led by Dr. Naoki Yahata (Fujita Health University School of Medicine) in collaboration with Dr. Yu-ichi Goto (National Center of Neurology and Psychiatry) and Dr. Ryuji Hata (Osaka Prefectural Hospital Organization).
Topics: Tools & Methods
