Chemically modified beta-glucuronidase crosses blood-brain barrier and clears neuronal storage in murine mucopolysaccharidosis VII

Abstract

Enzyme replacement therapy has been used successfully in many lysosomal storage diseases. However, correction of brain storage has been limited by the inability of infused enzyme to cross the blood-brain barrier. The newborn mouse is an exception because recombinant enzyme is delivered to neonatal brain after mannose 6-phosphate receptor-mediated transcytosis. Access to this route is very limited after 2 weeks of age. Recently, several studies showed that multiple infusions of high doses of enzyme partially cleared storage in adult brain. These results raised the question of whether correction of brain storage by repeated high doses of enzyme depends on mannose 6-phosphate receptor-mediated uptake or whether enzyme gains access to brain storage by another route when brain capillaries are exposed to prolonged, high levels of circulating enzyme. To address this question, we used an enzyme whose carbohydrate-dependent receptor-mediated uptake was inactivated by chemical modification. Treatment of human beta-glucuronidase (GUS) with sodium metaperiodate followed by sodium borohydride reduction (PerT-GUS) eliminated uptake by mannose 6-phosphate and mannose receptors in cultured cells and dramatically slowed its plasma clearance from a t(1/2) of <10 min to 18 h. Surprisingly, PerT-GUS infused weekly for 12 weeks was more effective in clearing central nervous system storage than native GUS at the same dose. In fact, PerT-GUS resulted in almost complete reversal of storage in neocortical and hippocampal neurons. This enhanced correction of neuronal storage by long-circulating enzyme, which targets no known receptor, suggests a delivery system across the blood-brain barrier that might be exploited therapeutically.

Fig. 1.

Stability of native GUS or PerT-GUS at 65°C. GUS or PerT-GUS was incubated at 65°C in heat-inactivation buffer, and aliquots were taken at 0, 30, 60, 120, and 180 min, cooled on ice, and then assayed for GUS activity. GUS (■) retained 90% of the initial activity after 180 min. PerT-GUS (○) was somewhat less stable but retained 40% of the original activity after 180 min.

Fig. 2.

Stability of native GUS or PerT-GUS in the lysosome after endocytosis by human fibroblasts. Human MPS VII fibroblasts were allowed to endocytose/pinocytose native GUS or PerT-GUS present in the normal growth medium in 35-mm culture dishes for 48 h at 37°C. After the indicated times, the cells were harvested and assayed for GUS activity. In fibroblasts, GUS (●) had a t½ of 18.9 days, whereas PerT-GUS (□) had a t½ of 12.9 days.

Fig. 3.

Clearance of infused GUS or PerT-GUS from the circulation. MPS VII mice were infused with GUS or PerT-GUS at a dose of 4 mg/kg of body weight. Blood samples were taken at the indicated times by supraorbital eye sticks into heparinized capillary tubes. After centrifugation, plasma was collected and assayed for GUS activity. GUS (●) was cleared from the circulation with a t½ of 11.7 min. Clearance of the PerT-GUS (□, ○, ▴) in three separate experiments was greatly prolonged with a t½ of 18.5 h.

Fig. 4.

Reduction in neuronal and meningeal storage with ERT with GUS and PerT-GUS. (A) Neocortical neurons from an untreated MPS VII mouse have abundant lysosomal storage in the cytoplasm (arrow). (B) After treatment with 4 mg/kg GUS, there is still a moderate amount of cytoplasmic storage (arrow) despite the therapy. (C) After 4 mg/kg PerT-GUS, there is a marked reduction in the amount of storage in the neocortical neurons (arrow). (D) The CA2 sector hippocampal neurons have abundant storage (arrow) in untreated MPS VII mice. (E) After treatment with GUS, the amount of storage in neurons (arrow) the same area of the hippocampus is similar to that of the untreated mouse. (F) After treatment with PerT-GUS, there is a remarkable reduction in the amount of storage in neurons (arrow) in the CA2 sector of the hippocampus. (G) The meninges of an untreated MPS VII mouse has abundant storage in fibroblasts around vessels (arrow). (H) Storage (arrow) is moderately decreased after treatment with GUS. (I) Treatment with PerT-GUS also produces moderate reduction in storage (arrow) in the meninges. (Scale bars: A–C, uranyl acetate–lead citrate, 10 μm; D–I, toluidine blue, 30 μm.)