In vivo adenine base editing of mutant Galc gene ameliorates Krabbe disease progression

Background: Krabbe disease (KD) is caused by mutation of the galactosylceramidase (GALC) gene, leading to deficient sphingolipid metabolism, which is essential for functional myelination. The twitcher (Galctwi/twi) mouse, a KD model with a premature termination codon (PTC) caused by a single-nucleotide G-to-A substitution at the 355th codon of the Galc gene, is a model candidate for treatment with adenine base editors (ABEs). ABEs have emerged exclusively among genome editing systems as viable therapeutic candidates to correct mutant genes. Methods: To confirm base editing efficiency of ABEs, mouse embryonic fibroblasts (MEFs) or mutant GALC HEK293T cells treated with three ABE variants (ABEmax, ABE8eWQ, ABE8e) were assessed using targeted deep sequencing. Each split-ABE8e vector was packaged into a dual-vector adeno-associated virus serotype 9 (AAV9) system and delivered to twitcher mice via intracerebroventricular injection on postnatal day 1. Thereafter, motor functions and survival rate were evaluated by rotarod test, clasping test and lifespan analysis. Various methods, including next-generation sequencing (NGS), qRT-PCR, enzyme activity assay, and flow cytometry, were used to measure the base correction rate of the target gene and verified restoration of GALC enzyme activity in the brain of ABE8e-treated mice. Additionally, myelin recovery was evaluated in the brain using histological analysis, magnetic resonance imaging (MRI), diffusion tensor imaging (DTI), and transmission electron microscopy (TEM). Results: The ABE8e-treated MEFs and mutant GALC HEK293T cells showed the most effective editing among the ABE variants tested. Three weeks after dual-AAV9 injection, the PTC was corrected in approximately 0.5% of genomic DNA and 5% of mRNA in twitcher mice. ABE8e treatment restored GALC enzymatic activity to approximately 5% of wild-type (WT) levels, while reducing the accumulation of psychosine—a major neurotoxic metabolite—by approximately 47% relative to WT. Moreover, histological analysis, TEM and, DTI and T2-weighted MRI showed preserved myelination and axonal integrity, along with amelioration of myelin deficits in the corpus callosum of ABE-treated twitcher mice. Five weeks after ABE8e administration, body weight recovered to approximately 64% of WT levels, accompanied by an extension of lifespan. In addition, clasping scores and rotarod performance improved to approximately 23% and 64% of WT levels, respectively. Conclusions: These data demonstrate a reliable application of base editing technology using ABEs as a potential treatment option for KD, progressing the development of therapeutics treating various genetic diseases. Graphical Abstract: Supplementary Information: The online version contains supplementary material available at 10.1186/s13073-026-01620-2.