Beth Levy

Enzyme replacement therapy for murine mucopolysaccharidosis type VII.

Recombinant mouse beta-glucuronidase administered intravenously to newborn mice with mucopolysaccharidosis type VII (MPS VII) is rapidly cleared from the circulation and localized in many tissues. Here we determine the tissue distribution of injected enzyme and describe its effects on the histopathology in 6-wk-old MPS VII mice that received either one injection of 28,000 U recombinant beta-glucuronidase at 5 wk of age or received six injections of 28,000 U given at weekly intervals beginning at birth. These mice were compared with untreated 6-wk-old MPS VII mice. The single injection decreased lysosomal distention in the fixed tissue macrophage system. MPS VII mice that received multiple injections had 27.8, 3.5, and 3.3% of normal levels of beta-glucuronidase in liver, spleen, and kidney, respectively. Brain had detectable beta-glucuronidase, ranging from 2.0-12.1% of normal. Secondary elevations of alpha-galactosidase and beta-hexosaminidase in brain, spleen, liver, and kidney were decreased compared with untreated MPS VII mice. Although no improvement was observed in chondrocytes, glia, and some neurons, the skeleton had less clinical and pathological evidence of disease and the brain had reduced lysosomal storage in meninges and selected neuronal groups. These data show that recombinant beta-glucuronidase treatment begun in newborn MPS VII mice provides enzyme to most tissues and significantly reduces or prevents the accumulation of lysosomal storage during the first 6 wk of life. Whether therapy begun later in life can achieve this level of correction remains to be established.

Murine mucopolysaccharidosis type VII: long term therapeutic effects of enzyme replacement and enzyme replacement followed by bone marrow transplantation.

We demonstrated previously that short term administration of recombinant beta-glucuronidase to newborn mice with mucopolysaccharidosis type VII reduced lysosomal storage in many tissues. Lysosomal storage accumulated gradually after cessation of enzyme replacement therapy. Mice alive at 1 yr of age had decreased bone deformities and less lysosomal storage in cortical neurons. Here we compare the effects of long term enzyme replacement initiated either at birth or at 6 wk of age, and of enzyme administration initiated at birth followed by syngeneic bone marrow transplantation (BMT) at 5 wk of age. Several mice from each treatment group lived to at least 1 yr of age. Liver and spleen samples had beta-glucuronidase levels ranging from 2.4 to 19.8% of normal and showed a parallel decrease in lysosomal storage. The combination of enzyme replacement therapy followed by BMT reduced lysosomal distension in meninges, corneal fibroblasts, and bone when compared with treatment with enzyme alone. Mice treated at birth had less lysosomal storage in some neurons of the brain and the skeletal dysplasia was less severe when compared to mice whose treatment was delayed until 6 wk of age. We conclude that both enzyme replacement alone and early enzyme replacement followed by BMT have long term positive effects on murine mucopolysaccharidosis type VII. In addition, treatment started at birth is far more effective than treatment initiated in young adults.

Enzyme replacement therapy for murine mucopolysaccharidosis type VII leads to improvements in behavior and auditory function.

Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is one of a group of lysosomal storage diseases that share many clinical features, including mental retardation and hearing loss. Lysosomal storage in neurons of the brain and the associated behavioral abnormalities characteristic of a murine model of MPS VII have not been shown to be corrected by either bone marrow transplantation or gene therapy. However, intravenous injections of recombinant beta-glucuronidase initiated at birth reduce the pathological evidence of disease in MPS VII mice. In this study we present evidence that enzyme replacement initiated at birth improved the behavioral performance and reduced hearing loss in MPS VII mice. Enzyme-treated MPS VII mice performed similarly to normal mice and significantly better than mock- treated MPS VII mice in every phase of the Morris Water Maze test. In addition, the auditory function of treated MPS VII mice was dramatically improved, and was indistinguishable from normal mice. These data indicate that some of the learning, memory, and hearing deficits can be prevented in MPS VII mice if enzyme replacement therapy is initiated early in life. These data also provide functional correlates to the biochemical and histopathological improvements observed after enzyme replacement therapy.

Enzyme replacement in murine mucopolysaccharidosis type VII: neuronal and glial response to beta-glucuronidase requires early initiation of enzyme replacement therapy.

We have previously shown that mucopolysaccharidosis type VII (MPS VII) mice receiving six weekly injections of recombinant β-glucuronidase from birth had improved cognitive ability and reduced central nervous system lysosomal storage. However, a single β-glucuronidase injection at 5 wk of age did not correct neuronal storage. We define the age at which central nervous system storage in MPS VII mice becomes resistant to β-glucuronidase therapy and determine the effect of enzyme on other tissues by comparing the histology of mice begun on therapy at various times after birth. MPS VII mice received injections on the day of birth and then weekly for 5 wk with 16 000U/g β-glucuronidase had reduced lysosomal storage in brain. The same therapy begun on d 14 of life or thereafter failed to correct neuronal storage, even when treatment was continued for six doses. Glial responsiveness or accessibility to enzyme also depended on early treatment. In contrast, leptomeningeal, osteoblast, and retinal pigment epithelial storage reduction depended on enzyme dose rather than age at initiation of therapy. Fixed tissue macrophage storage was reduced in all treated MPS VII mice, even those receiving a single dose. These observations indicate that fixed tissue macrophages in MPS VII mice remain sensitive to enzyme replacement therapy well into adulthood although neurons are responsive or accessible to enzyme therapy early in life. Because early initiation of enzyme replacement is important to achieve a central nervous system response, these studies emphasize the importance of newborn screening for lysosomal storage diseases so that early treatment can maximize the likelihood of a favorable therapeutic response.

Enzyme replacement with recombinant beta-glucuronidase in the newborn mucopolysaccharidosis type VII mouse.

ABSTRACT: β-Glucuronidase injected i.v. into newborn mucopolysaccharidosis VII mice was cleared from the circulation in less than 1 h and taken up by tissues in a distribution corresponding to the location of the mannose 6-phosphate receptor. One h after a 3.5-mg/kg β-glucuronidase injection, β-glucuronidase levels were equal to or greater than normal in every organ examined with the exception of the brain, where 31% normal activity was present. Enzyme was detectable histochemically in the major sites of pathology for mucopolysaccharidosis VII including bone, brain, heart, and fixed tissue macrophages. The half-life of recombinant β-glucuronidase activity in various organs of injected mucopolysaccharidosis VII mice was 1.5 to 4.5 d. These studies show that recombinant β-glucuronidase administered to newborn mice reaches the sites of clinically important storage in murine mucopolysaccharidosis VII.

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

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 β-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 t½ 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.

Enzyme replacement with recombinant beta-glucuronidase in murine mucopolysaccharidosis type VII: impact of therapy during the first six weeks of life on subsequent lysosomal storage, growth, and survival.

Treatment of mucopolysaccharidosis type VII (MPS VII) mice with recombinant mouse β-glucuronidase injections has been shown to deliver enzyme to most tissues and to reduce lysosomal storage during the first 6 wk of life. Here we determine the effect of enzyme therapy limited to the first 6 wk of life on survival and growth and follow the subsequent accumulation of lysosomal storage after β-glucuronidase treatment is discontinued. MPS VII mice received 28,000 U of β-glucuronidase i.v. at weekly intervals from birth to 6 wk of life and were killed at intervals up to 1 y after the last injection. By 29 d after the last enzyme injection, lysosomal storage material in bone was no different in amount than that seen in untreated MPS VII mice. By 85 d, the fixed tissue macrophage system, meninges, and brain glia had also accumulated storage comparable to that seen in untreated controls. One year after treatment, lysosomal storage was similar to that of untreated MPS VII mice in all sites except cortical neurons, where there was still a slight reduction. All treated mice that were not killed earlier, lived longer, were larger, and had milder facial and skeletal deformities than untreated MPS VII mice. These data show that enzyme replacement therapy in MPS VII mice during the first 6 wk of life improves survival and growth. After treatment is discontinued, storage accumulates slowly in the brain and more rapidly in the fixed tissue macrophage system. Whether therapy continued later in life can further improve survival and growth remains to be established.

Neuropathology of murine mucopolysaccharidosis type VII

Abstract We describe the neuropathology in mucopolysaccharidosis type VII (MPS VII) mice with a recessively inherited deficiency of the lysosomal enzyme β-glucuronidase. Affected animals have a shortened life span, are dysmorphic, dwarfed and have clinical evidence of behavioral and memory deficiencies. Widespread lysosomal distention with glycosaminoglycan accumulation affects most viscera. In the central nervous system there is progressive accumulation of lysosomal storage in neurons, glia and mesenchymal tissue. The morphological character and the amount of lysosomal storage varies among neuronal groups. In the hippocampus, regional variation in the abundance of lysosomal storage in the MPS VII mice correlates with regional variation in the amount of β-glucuronidase activity in normal mice. The MPS VII mouse provides a well-defined genetic system for the analysis of the neuropathology of MPS VII and is an attractive model on which to test the effects of potential therapies for lysosomal storage disease on the central nervous system.

Enzyme therapy in mannose receptor-null mucopolysaccharidosis VII mice defines roles for the mannose 6-phosphate and mannose receptors

Enzyme replacement therapy (ERT) is available for several lysosomal storage diseases. Except for Gaucher disease, for which an enzyme with exposed mannosyl residues targets mannose receptors (MR) on macrophages, ERT targets primarily the mannose 6-phosphate receptor (MPR). Most recombinant lysosomal enzymes contain oligosaccharides with both terminal mannosyl and mannose 6-phosphate residues. Effective MPR-mediated delivery may be compromised by rapid clearance of infused enzyme by the MR on fixed tissue macrophages, especially Kupffer cells. To evaluate the impact of this obstacle to ERT, we introduced the MR-null mutation onto the mucopolysaccharidosis type VII (MPS VII) background and produced doubly deficient MR−/− MPS VII mice. The availability of both MR+/+ and MR−/− mice allowed us to study the effects of eliminating the MR on MR- and MPR-mediated plasma clearance and tissue distribution of infused phosphorylated (P) and nonphosphorylated (NP) forms of human β-glucuronidase (GUS). In MR+/+ MPS VII mice, the MR clearance system predominated at doses up to 6.4 mg/kg P-GUS. Genetically eliminating the MR slowed plasma clearance of both P- and NP-GUS and enhanced the effectiveness of P-GUS in clearing storage in kidney, bone, and retina. Saturating the MR clearance system by high doses of enzyme also improved targeting to MPR-containing tissues such as muscle, kidney, heart, and hepatocytes. Although ablating the MR clearance system genetically is not practical clinically, blocking the MR-mediated clearance system with high doses of enzyme is feasible. This approach delivers a larger fraction of enzyme to MPR-expressing tissues, thus enhancing the effectiveness of MPR-targeted ERT.

Murine mucopolysaccharidosis type VII: the impact of therapies on the clinical course and pathology in a murine model of lysosomal storage disease.

Murine mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by a recessively inherited deficiency of the lysosomal enzyme β-glucuronidase. Affected mice have clinical, biochemical and pathological findings similar to those seen in humans with MPS VII (Sly syndrome), including growth retardation, facial dysmorphism, deafness, behavioural deficits and widespread glycosaminoglycan storage in lysosomes in the viscera, skeleton and brain. This mouse model is a useful tool for the evaluation of the effectiveness and experimental therapies for the MPS disorders. Syngeneic bone marrow transplantation performed in newborn MPS VII animals – before clinical evidence of disease is pronounced – prolongs life, improves hearing and bone growth, and prevents lysosomal storage in many sites, but does not correct the central nervous system disease. Enzyme therapy with β-glucuronidase from the first days of life does reduce lysosomal storage in the brain in murine MPS VII. The enzyme-replaced mice also have reduced visceral lysosomal storage, impressive normalization of their phenotype and an improved life span. The effectiveness of gene therapy for the treatment of lysosomal storage disease has also been tested using the MPS VII model. When transplanted into MPS VII mice, syngeneic haematopoietic stem cells or mouse skin fibroblasts infected with retrovirus expressing β-glucuronidase decreased storage, but only in the liver and spleen. Injection of an adenovirus vector expressing β-glucuronidase into the vitreous of the MPS VII mice reduced storage in the retinal pigment epithelium and corneal endothelium. Intravenous administration of the adenovirus vector transduced with the β-glucuronidase gene reduced liver and spleen storage and, when instilled into the cerebral ventricles, this viral vector caused β-glucuronidase production in epithelial cells lining the ventricles. Recently, retroviral vector-corrected MPS VII fibroblasts secreting high levels of β-glucuronidase were engrafted directly into the brains of adult MPS VII mice with resultant reduction in storage in neurons and glia adjacent to the grafts. Future efforts aimed at prolonging expression of the β-glucuronidase gene by viral vectors and more precisely directing the therapeutic effect to the skeleton and brain will be important in optimizing treatments for murine MPS VII and extending the results of such therapies to humans with MPS.