Mitochondrial Base Editing Enables Generation and Therapeutic Rescue of MT-ND4 Mutant Mouse Model of LHON

초록

Background: Leber Hereditary Optic Neuropathy (LHON) is a maternally inherited mitochondrial visual disorder caused mainly by mtDNA point mutations, most commonly MT-ND4 m.G11778A, leading to retinal ganglion cell (RGC) degeneration and progressive visual dysfunction. However, animal models carrying pathogenic mtDNA mutations have been lacking because CRISPR/Cas9 cannot be applicable to mtDNA editing due to the challenge of guide RNA delivery into mitochondria. This barrier has prevented the generation of LHON animal models carrying pathogenic mtDNA mutations, limiting in vivo studies of disease mechanisms and therapeutic evaluation. Recently developed CRISPR-free mtDNA base editors provide a new strategy to generate or correct pathogenic mitochondrial mutations in vivo. Aim: This study aimed to establish an human pathogenic mutation reproducing MT-ND4 mutant mouse model of LHON and to test whether in vivo mitochondrial base editing can correct the pathogenic mtDNA mutation and rescue retinal degeneration and visual dysfunction. Methods: High-fidelity DddA-derived cytosine base editors (Hifi-DdCBE) were used to generate the mouse MT-ND4 m.G11185A mutation, corresponding to the human LHON pathogenic m.G11778A variant. Retinal genotypes and phenotypes were examined using targeted deep sequencing, histology, optical coherence tomography, immunostaining for RGCs, optokinetic response, and electroretinography. To evaluate therapeutic potential, AAV2 expressing sTALED-V28R was delivered by intravitreal injection to correct the mtDNA mutation in retinal ganglion cells. Results: Canonical DddA11 induced widespread mitochondrial off-target mutations and embryonic arrest, whereas Hifi-DdCBE successfully generated MT-ND4 mutant mice with reduced off-target effects. These mice exhibited typical LHON phenotypes, including retina layer thinning, RGC degeneration, and visual dysfunction. Intravitreal injection of sTALED-V28R-AAV corrected mutant heteroplasmy in RGCs by approximately 36% on average, restored retinal thickness, increased RGC counts, and improved visual function. Conclusion: This study establishes MT-ND4 mutant mouse model of LHON and demonstrates that AAV-mediated in vivo mitochondrial base editing can correct pathogenic mtDNA mutations and rescue RGC degeneration and functional visual defects.