Mitochondrial Base Editing of the m.8993T>G Mutation Restores Bioenergetics and Neural Differentiation in Patient iPSCs

Point mutations in mitochondrial DNA (mtDNA) cause a range of neurometabolic disorders that currently have no curative treatments. The m.8993T>G mutation in the MT-ATP6 gene leads to neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) when heteroplasmy exceeds approximately 70 percent. We engineered a split DddA-derived cytosine base editor (DdCBE), each half fused to programmable TALE DNA-binding domains and a mitochondrial targeting sequence, to correct the m.8993T>G mutation in patient-derived induced pluripotent stem cells (iPSCs). Seven days after plasmid delivery, deep amplicon sequencing showed 35 ± 3 percent on-target C·G→T·A conversion at position 8993, reducing mutant heteroplasmy from 80 ± 2 percent to 45 ± 3 percent with less than 0.5 percent editing at ten predicted off-target loci. Edited cells exhibited a 25 percent increase in basal oxygen consumption rate, a 50 percent improvement in ATP-linked respiration, and a 2.3-fold restoration of ATP synthase activity. Directed neural differentiation yielded 85 ± 2 percent Nestin-positive progenitors compared to 60 ± 2 percent in unedited controls. Edits remained stable over 30 days in culture. These results establish mitochondrial base editing as a precise and durable strategy to ameliorate biochemical and cellular defects in NARP patient cells.