Synergistic Effects of Oct4-Induced Reprogramming and PE2max-Mediated Prime Editing on Myelination and Restoration of GALC activity in Krabbe disease

Abstract

ABSTRACT Krabbe disease (KD) is a fatal lysosomal storage disorder caused by a loss-of-function mutation in the galactosylceramidase (Galc) gene, resulting in impaired myelin metabolism and cytotoxic metabolite psychosine accumulation. This study aimed to investigate the therapeutic potential of a combinational approach that involves prime editing and in vivo cellular reprogramming in the KD twitcher mouse model. The prime editor PE2max was used to correct the point mutation in the Galc gene, and Oct4 overexpression was induced through intracerebroventricular injection of AAV9 on postnatal day 1 (PD1). Further, systemic delivery of AAVPHP.eB-PE2max was performed via facial vein injection. Behavioral assessments, including rotarod, hanging wire, cylinder, clasping, were conducted on PD21, PD28, and PD35. Animals were sacrificed on PD38, and subsequent molecular and histological analyses were conducted, including reverse quantitative transcription polymerase chain reaction, immunohistochemistry, electron microscopy, and magnetic resonance imaging, to assess Galc expression, remyelination (myelin basic protein), and cell fate markers such as Nestin, Olig2, Tuj1, and GFAP. BrdU labeling (PD7–9) confirmed that Oct4 induced neural precursor cell proliferation. The result revealed that Oct4 overexpression significantly increased the markers of the oligodendrocyte lineage and neural stem cell populations while reducing astrogliosis. Gene correction via PE2max restored the enzymatic activity of Galc and reduced psychosine accumulation. Combined treatment resulted in superior outcomes in myelin restoration, behavioral improvement, and extended survival compared with monotherapies and control groups. These results indicate that dual therapy combining Oct4-mediated reprogramming and PE2max-based gene correction represents a synergistic and promising therapeutic strategy for KD, addressing both cellular regeneration and the underlying enzymatic deficiency.