Abstract
Autosomal recessive mutations in the galactosidase β1 (GLB1) gene cause lysosomal β-gal deficiency, resulting in accumulation of galactose-containing substrates and onset of the progressive and fatal neurodegenerative lysosomal storage disease, GM1 gangliosidosis. Here, an enzyme replacement therapy (ERT) approach in fibroblasts from GM1 gangliosidosis patients with recombinant human β-gal (rhβ-gal) produced in Chinese hamster ovary cells enabled direct and precise rhβ-gal delivery to acidified lysosomes. A single, low dose (3 nm) of rhβ-gal was sufficient for normalizing β-gal activity and mediating substrate clearance for several weeks. We found that rhβ-gal uptake by the fibroblasts is dose-dependent and saturable and can be competitively inhibited by mannose 6-phosphate, suggesting cation-independent, mannose 6-phosphate receptor–mediated endocytosis from the cell surface. A single intracerebroventricularly (ICV) administered dose of rhβ-gal (100 μg) resulted in broad bilateral biodistribution of rhβ-gal to critical regions of pathology in a mouse model of GM1 gangliosidosis. Weekly ICV dosing of rhβ-gal for 8 weeks substantially reduced brain levels of ganglioside and oligosaccharide substrates and reversed well-established secondary neuropathology. Of note, unlike with the ERT approach, chronic lentivirus-mediated GLB1 overexpression in the GM1 gangliosidosis patient fibroblasts caused accumulation of a prelysosomal pool of β-gal, resulting in activation of the unfolded protein response and endoplasmic reticulum stress. This outcome was unsurprising in light of our in vitro biophysical findings for rhβ-gal, which include pH-dependent and concentration-dependent stability and dynamic self-association. Collectively, our results highlight that ICV-ERT is an effective therapeutic intervention for managing GM1 gangliosidosis potentially more safely than with gene therapy approaches.
Highlights
Autosomal recessive mutations in the galactosidase 1 (GLB1) gene cause lysosomal -gal deficiency, resulting in accumulation of galactose-containing substrates and onset of the progressive and fatal neurodegenerative lysosomal storage disease, GM1 gangliosidosis
Following cellular uptake of a single low dose of rh-gal (3 nM) for 18 h, lysosome-delivered rh-gal activity decays slowly with a half-life of ϳ9 days in GM1 gangliosidosis patient fibroblasts (Fig. 1F), which coincides with rapid turnover of GM1 ganglioside substrate (Fig. 1G and Fig. S3) and glycan substrates (Fig. 1H) within 1 week
Our results suggest that the biophysical properties of -gal, along with the therapeutic modality, should be considered when developing an effective treatment for GM1 gangliosidosis and that intermittent ICV-enzyme replacement therapy (ERT) dosing is a tunable therapeutic option that can safely and precisely deliver rh-gal to lysosomes to clear pathological lysosomal substrates and reverse neuropathology associated with the disease
Summary
A single low-nanomolar dose of purified rh-gal exhibits highly efficient CI-MPR–mediated cellular uptake in patient cells, which coincides with substrate clearance for several weeks. Chronic lentivirus-mediated WT GLB1 overexpression in GM1 gangliosidosis patient fibroblasts coincides with a timedependent increase in -gal activity (Fig. 7A) and -gal protein levels as detected by immunofluorescence in patient cells over 28 days (Fig. 7B; see Fig. 7C for quantification), which coincides with almost complete GM1 ganglioside substrate clearance (Fig. 7D; see Fig. 7E for quantification), suggestive of successful delivery of functional -gal to lysosomes. These results emphasize the potential for overexpressed -gal to be retained in the ER of GM1 gangliosidosis patient cells following lentivirus-mediated GLB1 gene therapy, where accumulation of precursor, nonlysosomal rh-gal can activate the unfolded protein response and trigger ER stress. Cellular uptake of rh-gal by CI-MPR–mediated endocytosis from the cell surface results in direct delivery of the enzyme to acidified lysosomes, where it remains as a stable mature dimer
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