Human GLB1 knockout cerebral organoids: A model system for testing AAV9-mediated GLB1 gene therapy for reducing GM1 ganglioside storage in GM1 gangliosidosis
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AuthorsLatour, Yvonne L.
Thomas, Sarah E.
Allende, Maria L.
Proia, Richard L.
Tifft, Cynthia J.
UMass Chan AffiliationsGene Therapy Center
Department of Neurology
Document TypeJournal Article
AAV serotype 9
bovine serum albumin
central nervous system
citrate phosphate buffer
green fluorescent protein
high-performance thin-layer chromatography
real-time quantitative polymerase chain reaction
human induced pluripotent stem cells
induced pluripotent stem
Amino Acids, Peptides, and Proteins
Congenital, Hereditary, and Neonatal Diseases and Abnormalities
Enzymes and Coenzymes
Genetics and Genomics
Nervous System Diseases
MetadataShow full item record
AbstractGM1 gangliosidosis is an autosomal recessive neurodegenerative disorder caused by the deficiency of lysosomal gangliosidebeta-galactosidase (beta-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 beta-gal deficiency. We screened for clones with < 5% of parental cell line beta-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 beta-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.
Mol Genet Metab Rep. 2019 Sep 11;21:100513. doi: 10.1016/j.ymgmr.2019.100513. eCollection 2019 Dec. Link to article on publisher's site
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/41206
RightsThis is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Except where otherwise noted, this item's license is described as This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).