DEVELOPMENT OF AN HEMATOPOIETIC STEM AND PROGENITOR CELLS GENE THERAPY FOR GM1 GANGLIOSIDOSIS

GM1-Gangliosidosis is a neurodegenerative lysosomal storage disorder (LSD) caused by mutations in the GLB1 gene, encoding the β-Galactosidase (β-Gal) lysosomal enzyme, and characterized by the accumulation of GM1 ganglioside in cells and tissues. The storage leads to severe neurodegeneration and premature death of the patients, occurring within the first few years of life in the most severe form of disease (infantile/ Type I). Currently there is no approved treatment for this condition. This study aimed at developing a safe and effective ex vivo gene therapy (GT) approach based on the use of hematopoietic stem and progenitor cells (HSPCs) and lentiviral vectors (LVs) expressing the GLB1 gene. We generated multiple LVs encoding either a codon-optimized human GLB1 cDNA (hu.GLB1.co-LV) or the murine isoform of the gene (mu.Glb1-LV), the latter demonstrating significantly higher enzymatic activity than the human homologue. Both vectors effectively restored β-Gal activity in vitro experiments in GM1 patient-derived fibroblasts, reduced pathological GM1-ganglioside storage, with potential for cross-correction of exposed cells, by enzyme secretion and uptake. The mu.Glb1-LV in particular, exhibited superior therapeutic potential, with a higher enzymatic activity per vector copy. We then developed a proof-of-concept study in a GM1 mouse model, i.e. Glb1-/- mice. We compared the hu.GLB1.co-LV (GT hu) and the mu.Glb1-LV (GT-mu) for murine HSPCs transduction and transplanted the transduced cells either by intravenous administration or by a combination of intravenous and intracerebroventricular administration (ICV) to enhance therapeutic efficacy. We also tested the potential effects of HSPC transplantation (HSCT) from wild-type mice, as a comparison. Overall, HSCT from wild-type donors did not result in significant benefit on the disease phenotype, while GT resulted in partial but significant restoration of β-Gal activity in the bone marrow, blood and brain, mitigation of neuromuscular symptoms and prolonged survival. In particular, mice receiving cells transduced with the mu.Glb1-LV displayed the greatest phenotypic effects, compared to mice receiving cells transduced with the hu.GLB1.co-LV, showing better performances in neuromotor tests and a greater reduction in GM1-Ganglioside storage in the brain, coupled to a minimal vector dose. Mice receiving HSPCs transduced with hu.GLB1.co-LV via IV+ICV showed a higher VCN in brain compared to mice receiving the same cells exclusively by IV administration. It may suggest a higher brain repopulation of HSPC-derived microglial-like cells by direct administration of HSPCs in the CNS. However, this higher VCN in the brain was not associated to a higher efficacy with respect to the IV only cell administration. Overall, these findings confirm GT has a superior therapeutic potential compared to HSCT, likely because of a higher expression of the therapeutic enzyme. Although in our study the treatment did not completely halt disease progression, the partial restoration of enzymatic activity and associated clinical benefits highlight the potential for further development. Future studies will focus on developing novel variants of the human enzyme which could combine a superior activity (equal or superior than murine isoform) and/or an improved stability to enhance the extent of metabolic cross correction and overall therapeutic potential of our approach.