J.H. Grubb, C. Vogler, W.S. Sly

Enzyme replacement therapy (ERT) is an established means of treating lysosomal storage diseases. Infused therapeutic enzymes are normally targeted to the lysosomes of affected cells by interactions with cell-surface receptors that recognize carbohydrate moieties, such as mannose and mannose 6-phosphate (M6P), on the enzymes. We have investigated alternative strategies to deliver lysosomal enzymes to lysosomes using the lysosomal enzyme B-glucuronidase (GUS) and the enzyme deficient Mucopolysaccharidosis Type VII (MPS VII) mouse model,

One strategy is a peptide based targeting system (GILT, Glycosylation- Independent Lysosomal Targeting). We took advantage of the interaction of a fragment of the insulin-like growth factor II (IGF-II) with the IGF-II binding site on the bifunctional, IGF-II/cation-dependent mannose 6-phosphate receptor. The chimeric GUS-IGF-II protein (GUS-GILT) was taken up by MPS VII fibroblasts in an M6P-independent manner and uptake was inhibited by IGF-II. Following ERT at an equivalent dose in the MPS VII mouse, GUS-GILT was more effective than the untagged enzyme at clearing lysosomal storage in glomerular podocytes and osteoblasts. In principle, the GILT-tag could be used to deliver any lysosomal enzyme or other protein of interest to the lysosome.

Correction of lysosomal storage in the brain has been limited by the inability of the infused enzyme to cross the blood-brain (BBB) barrier. This is based on data indicating that M6P receptor and mannose receptor uptake are blocked at the BBB. Recently we found that multiple high doses of GUS, over time could partially clear storage in the brain of the MPS VII mouse. Since both high dose and time were factors, we postulated that maintaining high levels of enzyme in the circulation for a protracted time was important. To address this question, we used an enzyme in which the carbohydrate-dependent receptor-mediated uptake was inactivated by chemical modification. Treatment of GUS with sodium meta-periodate followed by sodium borohydride reduction (PerT-GUS) inactivated the carbohydrate on the enzyme. This eliminated its uptake by M6P and mannose receptors in cultured cells and dramatically slowed its plasma clearance from a t½ of 10 minutes to 18 hours. When PerT-GUS was infused weekly for 12 weeks, it was found to be more effective in clearing central nervous system storage than native GUS at the same dose. Reversal of storage in neocortical and hippocampal neurons was almost complete. This strategy, which targets no known receptor, suggests a delivery system across the BBB that might be exploited therapeutically.

Another goal was to address prenatal storage which begins during prenatal life in patients with MPS. We hypothesized that Fc-tagged enzyme might deliver the missing enzyme across the placenta to the fetal circulation. It is known maternal IgG is transported across the placenta by the neonatal Fc-receptor (FcRn) which recognizes the Fc domain on IgG and and mediates transcytosis from maternal to fetal circulation. To test our hypothesis, we made a fusion protein (GUS-Fc) in which the IgG Fc domain was fused to the C-terminus of GUS. We observed that when infused into pregnant mothers on embryonic days 17 and 18, GUS-Fc was transported across the placenta, whereas untagged GUS was not. Plasma GUS-Fc levels in 1 day old mice previously treated in utero reached levels 100 times that of GUS levels in untreated wild type newborn mice. Reduced lysosomal storage in heart valves, liver and spleen provided evidence that in utero ERT with GUS-Fc, targeted sites of storage in the MPS VII fetus. This method could be used to deliver any lysosomal enzyme or protein of interest prenatally, to the fetus.

Keywords (Optional): 
lysosomal storage disease
enzyme replacement therapy