BMN 250, a fusion of lysosomal alpha-N-acetylglucosaminidase with IGF2, exhibits different patterns of cellular uptake into critical cell types of Sanfilippo syndrome B disease pathogenesis

Abstract

Sanfilippo syndrome type B (Sanfilippo B; Mucopolysaccharidosis type IIIB) occurs due to genetic deficiency of lysosomal alpha-N-acetylglucosaminidase (NAGLU) and subsequent lysosomal accumulation of heparan sulfate (HS), which coincides with devastating neurodegenerative disease. Because NAGLU expressed in Chinese hamster ovary cells is not mannose-6-phosphorylated, we developed an insulin-like growth factor 2 (IGF2)-tagged NAGLU molecule (BMN 250; tralesinidase alfa) that binds avidly to the IGF2 / cation-independent mannose 6-phosphate receptor (CI-MPR) for glycosylation independent lysosomal targeting. BMN 250 is currently being developed as an investigational enzyme replacement therapy for Sanfilippo B. Here we distinguish two cellular uptake mechanisms by which BMN 250 is targeted to lysosomes. In normal rodent-derived neurons and astrocytes, the majority of BMN250 uptake over 24 hours reaches saturation, which can be competitively inhibited with IGF2, suggestive of CI-MPR-mediated uptake. Kuptake, defined as the concentration of enzyme at half-maximal uptake, is 5 nM and 3 nM in neurons and astrocytes, with a maximal uptake capacity (Vmax) corresponding to 764 nmol/hr/mg and 5380 nmol/hr/mg, respectively. Similar to neurons and astrocytes, BMN 250 uptake in Sanfilippo B patient fibroblasts is predominantly CI-MPR-mediated, resulting in augmentation of NAGLU activity with doses of enzyme that fall well below the Kuptake (5 nM), which are sufficient to prevent HS accumulation. In contrast, uptake of the untagged recombinant human NAGLU (rhNAGLU) enzyme in neurons, astrocytes and fibroblasts is negligible at the same doses tested. In microglia, receptor-independent uptake, defined as enzyme uptake resistant to competition with excess IGF2, results in appreciable lysosomal delivery of BMN 250 and rhNAGLU (Vmax = 12,336 nmol/hr/mg and 5469 nmol/hr/mg, respectively). These results suggest that while receptor-independent mechanisms exist for lysosomal targeting of rhNAGLU in microglia, BMN 250, by its IGF2 tag moiety, confers increased CI-MPR-mediated lysosomal targeting to neurons and astrocytes, two additional critical cell types of Sanfilippo B disease pathogenesis.

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

“…7 A ). These patient cell–based proof-of-concept results are in agreement with our previous findings in Sanfilippo B patient fibroblasts ( 9 ), suggesting that long-lived lysosomal enzymes do not need to reach supraphysiological levels in order to be therapeutic and that enzyme activity only needs to be augmented above a critical threshold of ∼10–30% of normal lysosomal enzyme activity levels to mediate substrate turnover. Future dose-ranging and reaccumulation studies in the GLB1 KO mouse model will help to titrate the ICV-ERT dose and dosing frequency for rhβ-gal in clinical trials for GM1 gangliosidosis patients.…”

“…The entire range of clinical phenotypes for GM1 gangliosidosis, ranging from severe to relatively mild are clustered within a narrow range of residual lysosomal Beta-Gal activity, from 0-15% of normal control levels (8,9), suggesting that therapeutic strategies for this disease do not necessarily need to completely normalize Beta-Gal activity to mediate lysosomal degradation of stored substrates and prevent disease progression. In support of this we have previously demonstrated in patient cells that only very small increases in residual enzyme activity (~20% of normal enzyme activity levels) are sufficient to completely clear lysosomal storage material in a related lysosomal storage disease, Sanfilippo B syndrome (10).…”

Differential Uptake of NAGLU-IGF2 and Unmodified NAGLU in Cellular Models of Sanfilippo Syndrome Type B

“…Enzyme activity was determined using a 4MU (4-methylumbelliferyl- N -acetyl-α-glucosaminidase) synthetic fluorogenic substrate (EMD Millipore), according to a published protocol 3, 21, 22. To determine the K uptake , the concentration of enzyme mediating half-maximal uptake was measured.…”

The rapidly evolving view of lysosomal storage diseases

“…Intrathecal or intraventricular delivery of enzyme has been proposed for some mucopolysaccharidoses (Giugliani et al , 2018), metachromatic leukodystrophy (I Dali et al , 2020), and is now an approved treatment for neuronal ceroid lipofuscinosis 2, in which intraventricular infusions of cerliponase‐alpha reduced disease progression (Schulz et al , 2018). The use of chimeric lysosomal enzymes, in which the therapeutic enzyme is conjugated with different peptides or antibody components that exploit interactions with specific receptors and mediate transport across the blood–brain barrier, is also a strategy that is currently being investigated (Sonoda et al , 2018; Pardridge et al , 2018; Yogalingam et al , 2019).…”