An engineered galactosylceramidase construct improves AAV gene therapy for Krabbe disease in twitcher mice.

Krabbe disease is an inherited neurodegenerative disease caused by mutations in the galactosylceramidase gene. In the infantile form, patients die before 3 years of age. Systemic adeno-associated virus serotype 9 (AAV9) gene therapy was recently shown to reverse the disease course in human patients in another lethal infantile neurodegenerative disease. To explore AAV9 therapy for Krabbe disease, we engineered a codon-optimized AAV9 galactosylceramidase vector. We further incorporated features to allow AAV9-derived galactosylceramidase to more efficiently cross the blood-brain-barrier and be secreted from transduced cells. We tested the optimized vector by a single systemic injection in the twitcher mouse, an authentic Krabbe disease model. Untreated twitcher mice showed characteristic neuropathology and motion defects. They died prematurely with a median lifespan of 41 days. Intravenous injection in 2-day-old twitcher mice reduced central and peripheral neuropathology, significantly improved the gait pattern and body weight. Noticeably, the median lifespan was extended to 150 days. Intraperitoneal injection in 6 to 12-day-old twitcher mice also significantly improved the motor function, body weight, and median life span (to 104 days). Our results far exceed the ≤70 days median lifespan seen in all reported standalone systemic AAV therapies. Our study highlights the importance of vector engineering for Krabbe disease gene therapy. The engineered vector warrants further development.