The mitoDdCBE system as a mitochondrial gene therapy approach for pathological mtDNA mutations

Primary mitochondrial disorders are most often caused by deleterious mutations in the mtDNA. Here, we harnessed a mitochondrial base editor, mitoDdCBE, to introduce a compensatory edit (G5081A) in a mouse model that carries the pathological m.5024C-T mutation in the mitochondrial tRNAAla gene. For this, the mitoDdCBE gene construct was packaged in recombinant AAV9 and systemically injected into young mice. We found that the total tRNAAla levels, which are drastically reduced by the mutation, are restored by the m.5081G-A edit in a dose-dependent manner. However, an excessive expression of mitoDdCBE also induces extensive mtDNA off-target editing, counteracting this positive outcome. To address this, we successfully optimized the dosage to maximize the amount of compensatory edit generated with minimal off-target editing. These results show that mitochondrial base editors are promising candidates for gene therapy for mitochondrial disorders, but their overexpression need to be carefully controlled.