Abstract
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.