Prime editing via PE2max rescues galactosylceramidase function and neurobehavioral deterioration in Krabbe disease

Abstract

Krabbe disease (KD) is a life-threatening lysosomal storage disorder caused by mutations in the galactosylceramidase (GALC) gene, leading to psychosine accumulation, demyelination, and neurodegeneration in the central nervous system (CNS) and peripheral nervous system (PNS). Prime editing (PE) is a genome-editing method that allows for accurate base substitutions and small insertions or deletions without introducing double-strand DNA breaks. This study proposes a therapeutic strategy for KD using PE with the PE2max system to correct a point mutation and restore Galc function in Twitcher mice. Precise A-to-G correction at the Galc locus restored Galc expression and enzymatic activity, markedly enhancing molecular, histological, and behavioral measures. At postnatal day 1, PE2max was delivered via dual-adeno-associated virus (AAV) strategy: AAV-PHP.eB for CNS targeting and AAV-MaCPNS1 for PNS delivery. Deep genomic DNA and cDNA sequencing confirmed high editing fidelity with minimal off-target effects and background-level insertions/deletions (indels). CNS regions—including the frontal cortex, brainstem, and spinal cord—exhibited higher editing efficiency than other dissected regions, correlating with increased Galc mRNA expression and enzymatic activity. These effects were further supported by increased myelin basic protein expression, enhanced myelin integrity, and reduced globoid cell infiltration. Consistently, magnetic resonance imaging showed preserved white matter, and transmission electron microscopy revealed compact myelin and intact axons, indicating recovery at the macro- and ultrastructural levels. Although the extent of sciatic nerve editing was modest, the safety profile was excellent, with no observable toxicity in either wild-type or mutant mice. Our findings confirm that PE2max-mediated PE is a precise and safe in vivo genome-editing strategy that corrects disease-causing mutations without introducing double-strand breaks, supporting its therapeutic potential for monogenic neurodegenerative disorders.