Modeling GM1 gangliosidosis in human brain organoids to evaluate HSC-based gene therapy

Ganglioside monosialate 1 (GM1) gangliosidosis is a rare autosomal recessive lysosomal storage disorder (LSD) caused by mutations in the Galactosidase Beta 1 (GLB1) gene, leading to a deficiency of the lysosomal enzyme β-galactosidase (β-gal). Loss of enzymatic activity results in progressive accumulation of GM1 ganglioside within neurons and glial cells, triggering ER stress, neuroinflammation, and widespread neurodegeneration, ultimately leading to death in the most severe cases. Animal models only partially recapitulate human neuropathology and exhibit species-specific differences in β-gal functionality, highlighting the necessity for human-based systems to investigate disease mechanisms and test novel advanced therapeutic strategies. This thesis aims at establishing a human in vitro model of GM1 gangliosidosis using brain organoids (BOs) derived from patient-specific induced pluripotent stem cells (iPSCs) to evaluate the efficacy and molecular mechanism of hematopoietic stem cell (HSC)-based ex vivo gene therapies. Patient-specific iPSCs carrying GLB1 variants c.380G>A and c.481T>G, as well as wild-type (WT) iPSCs, were confirmed to be pluripotent through the expression of OCT4, SOX2, NANOG, OTX2, and TRA-1-60. β-gal activity, measured using the Mu-Gal assay, was detectable in WT cells but absent in GM1 fibroblasts and iPSCs, confirming their enzymatic deficiency, coherent with the genotype. These cell lines were differentiated into BOs. For GM1 iPSCs, optimized neural induction with extended dual-SMAD inhibition was required to promote pluripotency exit. Resulting neuroectodermal cysts expressed early neural markers (SOX1, PAX6, OTX2, N-cadherin), supporting forebrain BO generation from both WT and GM1 lines. Later analyses showed maturation into neuronal lineages belonging to forebrain. GM1 BOs exhibited absent β-gal activity and initial GM1 accumulation detected by cholera toxin B (CTxB) staining at 60 days of differentiation. To evaluate ex vivo gene therapy strategies, a BO-HSC co-culture system was established. Human GFP labelled CD34⁺ HSCs were co-cultured with 45-days-old BOs. Flow cytometry and immunostaining confirmed successful engraftment of GFP⁺/CD45⁺ cells and partial differentiation toward microglia-like cell types after 40 days, as indicated by IBA1 and PU.1 expression. Overall, this work establishes a physiologically relevant human model of GM1 gangliosidosis and opens the possibility to use it for investigating disease mechanisms and evaluating HSC–based gene therapies. The combination of GM1 patient-specific organoids with HSC-mediated enzyme delivery will provide a robust platform for preclinical assessment of cross correction capability of human HSC in a human setting.