Chemically modified β-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 ␤-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 t1 ͞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.deglycosylation ͉ enzyme replacement therapy ͉ hippocampal neurons ͉ lysosomal storage disease ͉ receptor-mediated endocytosis T he mucopolysaccharidosis (MPS) diseases, including MPS VII, are characterized by widespread storage of glycosaminoglycans in lysosomes in brain and viscera (1). Enzyme replacement therapy (ERT) has resulted in clearance of stored material from visceral organs and in clinical improvement in many patients with lysosomal storage diseases (LSDs) (2-5). Unfortunately, little infused enzyme crosses the blood-brain barrier (BBB), so limited improvement has been achieved in the central nervous system (CNS) (6).We have focused on improving delivery of enzyme to the brain by using ␤-glucuronidase (GUS) in the mouse model of MPS VII (Sly syndrome). When enzyme was infused into newborn mice, considerable enzyme was delivered to brain, and CNS storage was reduced (7-9). However, brain storage was resistant to clearance if ERT was begun after 2 weeks of age. Recent studies indicated that this enzyme delivery to the CNS in the newborn period was caused by mannose 6-phosphate receptor (M6PR)-mediated transcytosis (10). Down-regulation of this receptor by age 2 weeks appeared to explain the resistance of brain to ERT in the adult. More recently, however, we observed that multiple infusions of large doses ...

Chronic enzyme replacement therapy ameliorates neuropathology in alpha‐mannosidosis mice

“…This is in agreement with our previous studies16 showing that high enzyme doses are necessary to get sufficient amounts of enzyme into the CNS leading to a substantial reduction of brain storage when applied short term, suggesting a dose‐dependent uptake of the recombinant enzyme into the brain. Our studies are in agreement with other preclinical ERT studies in LSD animal models showing mostly beneficial effects of high‐dose injections of recombinant enzymes on primary lysosomal storage reduction and improvement of secondary pathological alterations such as neuroinflammation and behavioral deficits 29, 30, 31, 32, 33. Of note, in our preclinical study, the beneficial effect of ERT was most prominent for the 500 U/kg dosage but still significant at 125 U/kg for most of the parameters analyzed, suggesting that chronic treatment can at least to some extent compensate for lower enzyme activity.…”

“…According to Sly and coworkers using recombinant GUS in preclinical ERT studies with MPSVII mice,24, 34 mannose‐6 phosphate receptors (MPRs) that are responsible for intracellular lysosomal targeting of most lysosomes enzymes and their cellular uptake35, 36 cannot account for the uptake of recombinant lysosomal enzymes into adult brain due to their restricted expression in brain endothelial cells within the perinatal period (2 weeks after birth). In adult animals which lack expression of endothelial MPRs, administration of recombinant enzymes bearing mannose 6‐phosphate (M6P) residues might even diminish their uptake into brain as demonstrated for GUS 24, 30, 34. Of note, recombinant rhLAMAN which, in comparison with other lysosomal enzymes, is highly efficient in clearance of brain primary substrate storage is only marginally phosphorylated, suggesting primarily M6P‐independent uptake 16.…”

“…A prolonged circulation was suggested to enable the transfer of higher amounts of lysosomal enzymes into the brain. Therefore, deglycosylation or chemical modification was used to increase the circulatory half‐life of lysosomal enzymes such as Tripeptidyl peptidase type 1 (TTP1)39 and GUS 30, 40. However, in contrast to GUS, glycan modification of TTP1 did not improve its targeting to brain.…”

Transport of Arylsulfatase A across the Blood-Brain Barrier in Vitro

“…Sly and co-workers (17,27) have analyzed blood-to-brain transfer of the lysosomal enzyme ␤-glucuronidase in a mouse model of mucopolysaccharidosis type VII treated by ERT. The authors found that overexpression of MPR300 at the BBB increases cerebral ␤-glucuronidase levels and glycosaminoglycan clearance from brain cells (27).…”

“…Exposed carbohydrates of rhASA were inactivated by sodium metaperiodate treatment (17). One milligram of rhASA was incubated with a final concentration of 10 mM sodium metaperiodate in 100 mM NaCl/20 mM sodium phosphate (pH 6.0) for 30 min at 20°C.…”