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Intracerebroventricular enzyme replacement therapy with ?-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.

ABSTRACT: 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. Here, an enzyme replacement therapy (ERT) approach in fibroblasts from GM1 gangliosidosis patients with recombinant human ?-gal (rh?-gal) produced in Chinese hamster ovary cells enabled direct and precise rh?-gal delivery to acidified lysosomes. A single, low dose (3 nm) of rh?-gal was sufficient for normalizing ?-gal activity and mediating substrate clearance for several weeks. We found that rh?-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 rh?-gal (100 ?g) resulted in broad bilateral biodistribution of rh?-gal to critical regions of pathology in a mouse model of GM1 gangliosidosis. Weekly ICV dosing of rh?-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 ?-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 rh?-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.

SUBMITTER: Chen JC

PROVIDER: S-EPMC7521651 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Intracerebroventricular enzyme replacement therapy with β-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.

Chen Joseph C JC Luu Amanda R AR Wise Nathan N Angelis Rolando De R Agrawal Vishal V Mangini Linley L Vincelette Jon J Handyside Britta B Sterling Harry H Lo Melanie J MJ Wong Hio H Galicia Nicole N Pacheco Glenn G Van Vleet Jeremy J Giaramita Alexander A Fong Sylvia S Roy Sushmita M SM Hague Chuck C Lawrence Roger R Bullens Sherry S Christianson Terri M TM d'Azzo Alessandra A Crawford Brett E BE Bunting Stuart S LeBowitz Jonathan H JH Yogalingam Gouri G

The Journal of biological chemistry 20190903 39

Autosomal recessive mutations in the galactosidase β1 (<i>GLB1</i>) 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 aci ...[more]

PMID: 31481471

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Project description:GM1-gangliosidosis is a lysosomal disease resulting from a deficiency in the hydrolase β-galactosidase (β-gal) and subsequent accumulation of gangliosides, primarily in neuronal tissue, leading to progressive neurological deterioration and eventually early death. Lysosomal diseases with neurological involvement have limited non-invasive therapies due to the inability of lysosomal enzymes to cross the blood-brain barrier (BBB). A novel fusion enzyme, labeled mTfR-GLB1, was designed to act as a ferry across the BBB by fusing β-gal to the mouse monoclonal antibody against the mouse transferrin receptor and tested in a murine model of GM1-gangliosidosis (β-gal-/-). Twelve hours following a single intravenous dose of mTfR-GLB1 (5.0 mg/kg) into adult β-gal-/- mice showed clearance of enzyme activity in the plasma and an increase in β-gal enzyme activity in the liver and spleen. Long-term efficacy of mTfR-GLB1 was assessed by treating β-gal-/- mice intravenously twice a week with a low (2.5 mg/kg) or high (5.0 mg/kg) dose of mTfR-GLB1 for 17 weeks. Long-term studies showed high dose mice gained weight normally compared to vehicle-treated β-gal-/- mice, which are significantly heavier than heterozygous controls. Behavioral assessment at six months of age using the pole test showed β-gal-/- mice treated with mTfR-GLB1 had improved motor function. Biochemical analysis showed an increase in β-gal enzyme activity in the high dose group from negligible levels to 20% and 11% of heterozygous levels in the liver and spleen, respectively. Together, these data show that mTfR-GLB1 is a catalytically active β-gal fusion enzyme in vivo that is readily taken up into tissues. Despite these indications of bioactivity, behavior tests other than the pole test, including the Barnes maze, inverted screen, and accelerating rotarod, showed limited or no improvement of treated mice compared to β-gal-/- mice receiving vehicle only. Further, administration of mTfR-GLB1 was insufficient to create measurable increases in β-gal enzyme activity in the brain or reduce ganglioside content (biochemically and morphologically).

Project description:Lysosomal beta-galactosidase (beta-Gal) occurs either alone in monomeric and dimeric forms, or in a high-M(r) complex with at least two additional proteins. One is neuraminidase and the second is the protective protein, which has also been shown to possess carboxypeptidase activity. beta-Gal activity is deficient in GM1-gangliosidosis as a primary defect, and is secondarily affected in galactosialidosis (GS), where the primary defect is the absence of protective protein activity. Fibroblasts from three patients with GM1-gangliosidosis, type 1, showed markedly reduced amounts of beta-Gal cross-reacting material (CRM), and a fourth appeared to have normal levels. A patient with type 2 GM1-gangliosidosis was also found to be CRM-normal. These findings demonstrate that patients with GM1-gangliosidosis type 1 are heterogeneous with respect to the level of residual beta-Gal protein. Fibroblasts from four patients with GS were strongly CRM-positive with an anti-beta-Gal antibody, as was a sample of brain from one of these patients, suggesting that the loss of beta-Gal activity is linked to a subtler change in the primary structure of the enzyme than has been previously thought. While three GS cell lines displayed reduced carboxypeptidase activity (to 32-42% of the control), one cell line was completely devoid of activity, demonstrating that while carboxypeptidase activity is a property of the protective protein this action is distinct and separate from its protective role. On direct immunoprecipitation with anti-beta-Gal antibody, a portion of the total carboxypeptidase activity co-precipitated with beta-Gal from extracts of normal and GM1-gangliosidosis cells, consistent with the presence of the complex in these cells. However, no carboxypeptidase activity was precipitable with this antibody from GS fibroblasts, suggesting the absence of complex from these cells. To examine this further, the various forms of beta-Gal were resolved by h.p.l.c. molecular-sieve chromatography. Three forms of beta-Gal activity were resolved in normal cells: a complex, a dimer and a monomer. Residual beta-Gal activity of GS cells resolved into two of these forms, the complex and the monomer. In normal and GM1-gangliosidosis cells a portion of the total carboxypeptidase activity co-chromatographed with the complex while the bulk of the activity occurred in a single 36,000-M(r) peak. Only the low-M(r) carboxypeptidase activity was detected in GS cells. This confirms our results on immunoprecipitation indicating that portions of the beta-Gal and the carboxypeptidase activities exist outside the complex in normal, GM1-gangliosidosis and GS cells. In summary, the loss of protective protein function from GS cells results in disproportionate loss of the dimeric and monomeric forms of beta-Gal activity, but does not result in the complete degradation of the protein.

Dataset Information

Intracerebroventricular enzyme replacement therapy with ?-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.

Publications

Intracerebroventricular enzyme replacement therapy with β-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.

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