GM1 gangliosidosis is an autosomal recessive
neurodegenerative disorder caused by the deficiency of lysosomal β-
galactosidase (β-gal) and resulting in accumulation of
GM1 ganglioside. The disease spectrum ranges from infantile to late onset and is uniformly fatal, with no effective
therapy currently available. Although animal models have been useful for understanding disease pathogenesis and exploring therapeutic targets, no relevant human central nervous system (CNS) model system has been available to study its early pathogenic events or test
therapies. To develop a model of human
GM1 gangliosidosis in the CNS, we employed CRISPR/Cas9 genome editing to target GLB1 exons 2 and 6, common sites for mutations in patients, to create isogenic induced pluripotent stem (iPS) cell lines with lysosomal β-gal deficiency. We screened for clones with <5% of parental cell line β-gal
enzyme activity and confirmed GLB1 knockout clones using
DNA sequencing. We then generated GLB1 knockout cerebral organoids from one of these GLB1 knockout iPS cell clones. Analysis of GLB1 knockout organoids in culture revealed progressive accumulation of
GM1 ganglioside. GLB1 knockout organoids microinjected with AAV9-GLB1 vector showed a significant increase in β-gal activity and a significant reduction in
GM1 ganglioside content compared with AAV9-GFP-injected organoids, demonstrating the efficacy of an AAV9 gene therapy-based approach in
GM1 gangliosidosis. This proof-of-concept in a human cerebral organoid model completes the pre-clinical studies to advance to clinical trials using the AAV9-GLB1 vector.