Mucopolysaccharidosis I, II, and VI: Brief review and guidelines for treatment

Mucopolysaccharidoses (MPS) are rare genetic diseases caused by the deficiency of one of the lysosomal enzymes involved in the glycosaminoglycan (GAG) breakdown pathway. This metabolic block leads to the accumulation of GAG in various organs and tissues of the affected patients, resulting in a multisystemic clinical picture, sometimes including cognitive impairment. Until the beginning of the XXI century, treatment was mainly supportive. Bone marrow transplantation improved the natural course of the disease in some types of MPS, but the morbidity and mortality restricted its use to selected cases. The identification of the genes involved, the new molecular biology tools and the availability of animal models made it possible to develop specific enzyme replacement therapies (ERT) for these diseases. At present, a great number of Brazilian medical centers from all regions of the country have experience with ERT for MPS I, II, and VI, acquired not only through patient treatment but also in clinical trials. Taking the three types of MPS together, over 200 patients have been treated with ERT in our country. This document summarizes the experience of the professionals involved, along with the data available in the international literature, bringing together and harmonizing the information available on the management of these severe and progressive diseases, thus disclosing new prospects for Brazilian patients affected by these conditions.     

Prolonged expression of a lysosomal enzyme in mouse liver after Sleeping Beauty transposon-mediated gene delivery: implications for non-viral gene therapy of mucopolysaccharidoses

BACKGROUND: The Sleeping Beauty (SB) transposon system is a non-viral vector system that can integrate precise sequences into chromosomes. We evaluated the SB transposon system as a tool for gene therapy of mucopolysaccharidosis (MPS) types I and VII. METHODS: We constructed SB transposon plasmids for high-level expression of human beta-glucuronidase (hGUSB) or alpha-L-iduronidase (hIDUA). Plasmids were delivered with and without SB transposase to mouse liver by rapid, high-volume tail-vein injection. We studied the duration of expressed therapeutic enzyme activity, transgene presence by PCR, lysosomal pathology by toluidine blue staining and cell-mediated immune response histologically and by immunohistochemical staining. RESULTS: Transgene frequency, distribution of transgene and enzyme expression in liver and the level of transgenic enzyme required for amelioration of lysosomal pathology were estimated in MPS I and VII mice. Without immunomodulation, initial GUSB and IDUA activities in plasma reached > 100-fold of wild-type (WT) levels but fell to background within 4 weeks post-injection. In immunomodulated transposon-treated MPS I mice plasma IDUA persisted for over 3 months at up to 100-fold WT activity in one-third of MPS I mice, which was sufficient to reverse lysosomal pathology in the liver and, partially, in distant organs. Histological and immunohistochemical examination of liver sections in IDUA transposon-treated WT mice revealed inflammation 10 days post-injection consisting predominantly of mononuclear cells, some of which were CD4- or CD8-positive. CONCLUSIONS: Our results demonstrate the feasibility of achieving prolonged expression of lysosomal enzymes in the liver and reversing MPS disease in adult mice with a single dose of therapeutic SB transposons.     

Mucopolysaccharidoses in northern Brazil: Targeted mutation screening and urinary glycosaminoglycan excretion in patients undergoing enzyme replacement therapy

Mucopolysaccharidoses (MPS) are rare lysosomal disorders caused by the deficiency of specific lysosomal enzymes responsible for glycosaminoglycan (GAG) degradation. Enzyme Replacement Therapy (ERT) has been shown to reduce accumulation and urinary excretion of GAG, and to improve some of the patients' clinical signs. We studied biochemical and molecular characteristics of nine MPS patients (two MPS I, four MPS II and three MPS VI) undergoing ERT in northern Brazil. The responsiveness of ERT was evaluated through urinary GAG excretion measurements. Patients were screened for eight common MPS mutations, using PCR, restriction enzyme tests and direct sequencing. Two MPS I patients had the previously reported mutation p.P533R. In the MPS II patients, mutation analysis identified the mutation p.R468W, and in the MPS VI patients, polymorphisms p.V358M and p.V376M were also found. After 48 weeks of ERT, biochemical analysis showed a significantly decreased total urinary GAG excretion in patients with MPS I (p < 0.01) and MPS VI (p < 0.01). Our findings demonstrate the effect of ERT on urinary GAG excretion and suggest the adoption of a screening strategy for genotyping MPS patients living far from the main reference centers.     

Enzyme Replacement in a Human Model of Mucopolysaccharidosis IVA In Vitro and Its Biodistribution in the Cartilage of Wild Type Mice

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active (∼2 U/mg) and pure (≥97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (K_uptake = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA.     

Glycosaminoglycan Storage in Cultured Neonatal Murine Mucopolysaccharidosis Type VII Neuroglial Cells and Correction by β-Glucuronidase Gene Transfer

Abstract: The inherited deficiency of β-glucuronidase activity causes the lysosomal storage disorder mucopolysaccharidosis (MPS) type VII (Sly disease). The sequential catabolism of glycosaminoglycans in lysosomes is blocked, and undegraded substrates accumulate in cells of many tissues, including neurons and glia in the brain. To evaluate the deficient metabolic pathway, primary cultures of mixed brain cells were established from newborn MPS VII mice. β-Glucuronidase levels and glycosaminoglycan accumulation were studied in normal, carrier, and MPS VII cells. Retroviral vector-mediated transfer of a normal β-glucuronidase cDNA corrected the enzymatic deficiency in MPS VII cells and restored glycosaminoglycan catabolism to normal. High levels of β-glucuronidase expression were sustained in vector-corrected nondividing glial cell cultures for >2 months. These studies provide an in vitro model for evaluating somatic gene transfer in neural cells affected in mucopolysaccharidoses.     

Plasmatic kinetics of dermatan sulfate during enzyme replacement therapy with iduronate-2-sulfatase in a mucopolysaccharidosis II Patient

Enzyme replacement therapy (ERT) is the worldwide standard of care for a number of mucopolysaccharidosis (MPS) diseases. We report a kinetic study of plasmatic dermatan sulfate (DS) in a 3-year-old subject affected by a severe form of MPS II during the first 10 months of ERT with Idursulfase. A strong increase in the DS plasmatic concentration was measured immediately after the first enzyme infusion, with a maximum after 3 h, followed by a continuous decrease in the 8–15 days following the beginning of treatment. After this, a constant plasmatic content of DS concentration was observed. Overall, during the 10-month treatment period, ERT reduced the plasmatic concentration of DS up to ~80–85 %, but it was unable to totally remove it from the blood. We can suppose that immediately after the first enzyme administrations, a large amount of abnormal DS is removed from tissues reaching the blood compartment and eliminated via the urine, and thereafter only minimal changes are observed. The persistency of the residual amounts of DS with the actually recommended dosage in our Patient may suggest the opportunity to promote further studies with increased enzyme dosages to completely remove the accumulation of lysosomal DS.     

Adipose deficiency and aberrant autophagy in a Drosophila model of MPS VII is corrected by pharmacological stimulators of mTOR

Mucopolysaccharidosis type VII (MPS VII) is a recessively inherited lysosomal storage disorder caused due to β-glucuronidase (β-GUS) enzyme deficiency. Prominent clinical symptoms include hydrops fetalis, musculoskeletal deformities, neurodegeneration and hepatosplenomegaly leading to premature death in most cases. Apart from these, MPS VII is also characterized as adipose storage deficiency disorder although the underlying mechanism of this lean phenotype in the patients or β-GUS-deficient mice still remains a mystery. We addressed this issue using our recently developed Drosophila model of MPS VII (the CG2135^-/- fly), which also exhibited a significant loss of body fat. We report here that the lean phenotype of the CG2135^?/? larvae is due to fewer number of adipocytes, smaller lipid droplets and reduced adipogenesis. Our data further revealed that there is an abnormal accumulation of autophagosomes in the CG2135^?/? larvae due to autophagosome-lysosome fusion defect. Decreased lysosome-mediated turnover also led to attenuated mTOR activity in the CG2135^?/? larvae. Interestingly, treatment of the CG2135^?/? larvae with mTOR stimulators, 3BDO or glucose, led to the restoration of mTOR activity with simultaneous correction of the autophagy defect and adipose storage deficiency. Our finding thus established a hitherto unknown mechanistic link between autophagy dysfunction, mTOR downregulation and reduced adiposity in MPS VII.     

Lysosomal leukocyte beta-D-glucuronidase during enzyme replacement therapy in Fabry disease

OBJECTIVE: Fabry disease results from a deficiency in the activity of alpha-d-galactosidase A and subsequent accumulation of neutral glycosphingolipids in lysosomes. This study investigated whether lysosomal enzymes can indicate biochemical changes in the lysosomal apparatus induced by enzyme replacement therapy (ERT). DESIGN AND METHODS: Eight patients were monitored by clinical and biochemical tests and several lysosomal glycohydrolases were measured in plasma and leucocytes. RESULTS: Before starting ERT, beta-d-glucuronidase in leukocytes was markedly increased. After 1 month of therapy, enzyme levels dropped in all patients. In the patients who regularly followed the therapy, the enzyme levels remained stable for the next 20 months. In one patient who interrupted therapy for 2 months, the enzyme levels rose again. CONCLUSIONS: Lysosomal enzymes can be useful for monitoring biochemical changes in patients with Fabry disease receiving ERT. Though these findings refer to only a small number of patients, the correlation between beta-d-glucuronidase levels and ERT is interesting and might serve as a basis for further studies to define the potential of this enzyme in monitoring the effects of ERT in lysosomal storage disorders.     

Substrate reduction augments the efficacy of enzyme therapy in a mouse model of Fabry disease

Fabry disease is an X-linked glycosphingolipid storage disorder caused by a deficiency in the activity of the lysosomal hydrolase α-galactosidase A (α-gal). This deficiency results in accumulation of the glycosphingolipid globotriaosylceramide (GL-3) in lysosomes. Endothelial cell storage of GL-3 frequently leads to kidney dysfunction, cardiac and cerebrovascular disease. The current treatment for Fabry disease is through infusions of recombinant α-gal (enzyme-replacement therapy; ERT). Although ERT can markedly reduce the lysosomal burden of GL-3 in endothelial cells, variability is seen in the clearance from several other cell types. This suggests that alternative and adjuvant therapies may be desirable. Use of glucosylceramide synthase inhibitors to abate the biosynthesis of glycosphingolipids (substrate reduction therapy, SRT) has been shown to be effective at reducing substrate levels in the related glycosphingolipidosis, Gaucher disease. Here, we show that such an inhibitor (eliglustat tartrate, Genz-112638) was effective at lowering GL-3 accumulation in a mouse model of Fabry disease. Relative efficacy of SRT and ERT at reducing GL-3 levels in Fabry mouse tissues differed with SRT being more effective in the kidney, and ERT more efficacious in the heart and liver. Combination therapy with ERT and SRT provided the most complete clearance of GL-3 from all the tissues. Furthermore, treatment normalized urine volume and uromodulin levels and significantly delayed the loss of a nociceptive response. The differential efficacies of SRT and ERT in the different tissues indicate that the combination approach is both additive and complementary suggesting the possibility of an improved therapeutic paradigm in the management of Fabry disease.     

Enhanced response to enzyme replacement therapy in Pompe disease after the induction of immune tolerance

Pompe disease, which results from mutations in the gene encoding the glycogen-degrading lysosomal enzyme acid alpha -glucosidase (GAA) (also called "acid maltase"), causes death in early childhood related to glycogen accumulation in striated muscle and an accompanying infantile-onset cardiomyopathy. The efficacy of enzyme replacement therapy (ERT) with recombinant human GAA was demonstrated during clinical trials that prolonged subjects' overall survival, prolonged ventilator-free survival, and also improved cardiomyopathy, which led to broad-label approval by the U.S. Food and Drug Administration. Patients who lack any residual GAA expression and are deemed negative for cross-reacting immunologic material (CRIM) have a poor response to ERT. We previously showed that gene therapy with an adeno-associated virus (AAV) vector containing a liver-specific promoter elevated the GAA activity in plasma and prevented anti-GAA antibody formation in immunocompetent GAA-knockout mice for 18 wk, predicting that liver-specific expression of human GAA with the AAV vector would induce immune tolerance and enhance the efficacy of ERT. In this study, a very low number of AAV vector particles was administered before initiation of ERT, to prevent the antibody response in GAA-knockout mice. A robust antibody response was provoked in naive GAA-knockout mice by 6 wk after a challenge with human GAA and Freund's adjuvant; in contrast, administration of the AAV vector before the GAA challenge prevented the antibody response. Most compellingly, the antibody response was prevented by AAV vector administration during the 12 wk of ERT, and the efficacy of ERT was thereby enhanced. Thus, AAV vector-mediated gene therapy induced a tolerance to introduced GAA, and this strategy could enhance the efficacy of ERT in CRIM-negative patients with Pompe disease and in patients with other lysosomal storage diseases.     

Bone, joint and tooth development in mucopolysaccharidoses: relevance to therapeutic options

The mucopolysaccharidoses (MPS) are prominent among the lysosomal storage diseases. The intra-lysosomal accumulation of glycosaminoglycans (GAGs) in this group of diseases, which are caused by several different enzyme deficiencies, induces a cascade of responses that affect cellular functions and maintenance of the extra-cellular matrix. Against the background of normal tissue-specific processes, this review summarizes and discusses the histological and biochemical abnormalities reported in the bones, joints, teeth and extracellular matrix of MPS patients and animal models. With an eye to the possibilities and limitations of reversing the pathological changes in the various tissues, we address therapeutic challenges, and present a model in which the cascade of pathologic events is depicted in terms of primary and secondary events.     

Rapid prenatal testing for human beta-glucuronidase deficiency (MPS VII)

Prenatal diagnosis for the lysosomal storage disorders is typically achieved by enzymatic analysis of the relevant lysosomal enzyme in cultured amniocytes or chorionic villi. While prenatal diagnosis of some genetic diseases can be done by analysis of pertinent metabolites in amniotic fluid, there are few data regarding prenatal diagnosis of lysosomal disorders by enzyme analysis of amniotic fluid. Prenatal diagnosis by enzyme analysis of amniotic fluid has the potential advantage of providing a more rapid prenatal test result. In this study we describe an assay for the prenatal diagnosis of the mucopolysaccharidosis beta-glucuronidase deficiency (MPS VII; MIM #253220) using amniotic fluid and we confirm its reliability in detecting an affected fetus in an at-risk pregnancy by enzyme analysis of cultured amniocytes and fetal fibroblasts. Because MPS VII is rare and few instances of prenatal diagnosis for this and nearly all other lysosomal disorders have been accomplished by enzyme analysis of amniotic fluid, confirmation of results obtained from enzyme analysis of amniotic fluid should be carried out by enzyme or mutation analysis using cultured amniocytes or chorionic villus specimens.     

Systemic and central nervous system correction of lysosomal storage in mucopolysaccharidosis type VII mice

Mucopolysaccharidosis (MPS) type VII patients lack functional beta-glucuronidase, leading to systemic and central nervous system dysfunction. In this study we tested whether recombinant adenovirus that encodes beta-glucuronidase (Adbetagluc), delivered intravenously and into the brain parenchyma of MPS type VII mice, could provide long-term transgene expression and correction of lysosomal distension. We also tested whether systemic treatment with the immunosuppressive anti-CD40 ligand antibody, MR-1, affected transgene expression. We found substantial plasma beta-glucuronidase activity for over 9 weeks after gene transfer in the MR-1- treated group, with subsequent decline in activity corresponding to a delayed anti-beta-glucuronidase antibody response. At 16 weeks, near wild-type amounts of beta-glucuronidase activity and striking reduction of lysosomal pathology were detected in livers from mice that had received either MR-1 cotreatment or control antibody. In the lung and kidney, beta-glucuronidase activity was markedly higher for the MR-1-treated group. beta-Glucuronidase activity in the brain persisted independently of MR-1 treatment. Activity was intense in the injected hemisphere and was also evident in the noninjected cortex and striatum, with dramatic improvements in storage deposits in areas of both hemispheres. These results indicate that prolonged enzyme expression from transgenes delivered to deficient liver and brain can mediate pervasive correction and illustrate the potential for gene therapy of MPS and other lysosomal storage diseases.     

Common antigenicity for two glycosidases

Enzyme replacement therapy (ERT) has proven to be an effective therapy for some lysosomal storage disorder (LSD) patients. A potential complication during ERT is the generation of an immune response against the replacement protein. We have investigated the antigenicity of two distantly related glycosidases, alpha-glucosidase (Pompe disease or glycogen storage disease type II, GSD II), and alpha-L-iduronidase (Hurler syndrome, mucopolysaccharidosis type I, MPS I). The linear sequence epitope reactivity of affinity purified polyclonal antibodies to recombinant human alpha-glucosidase and alpha-L-iduronidase was defined, to both glycosidases. The polyclonal antibodies exhibited some cross-reactive epitopes on the two proteins. Moreover, a monoclonal antibody to the active site of alpha-glucosidase showed cross-reactivity with a catalytic structural element of alpha-L-iduronidase. In a previous study, in MPS I patients who developed an immune response to ERT, this same site on alpha-L-iduronidase was highly antigenic and the last to tolerise following repeated enzyme infusions. We conclude that glycosidases can exhibit cross-reactive epitopes, and infer that this may relate to common structural elements associated with their active sites.     

Low β-glucuronidase enzyme activity and mutations in the human β-glucuronidase gene in mild mucopolysaccharidosis type VII, pseudodeficiency and a heterozygote

Deficiency of β-glucuronidase is the cause of the human lysosomal storage disorder mucopolysaccharidosis type VII (MPS VII). The wide interfamilial variation in the presentation of this disorder complicates clinical diagnosis. Since greatly reduced β-glucuronidase enzyme activity may also be found in healthy individuals (pseudodeficiency), diagnosis based on the biochemical phenotype is also difficult. This is illustrated by the patients studied here, who had extremely mild symptoms confined to the spine, or tachycardia, or upper respiratory infection, and who had low β-glucuronidase activity, and excessive granulation of granulocytes and monocytes on routine blood smears. Low enzyme activity was caused by mutations in the β-glucuronidase gene in all cases. One patient was homozygous for the previously described D152N allele. Family information and ³⁵SO₄-uptake studies clearly demonstrated that he was pseudodeficient, with symptoms unrelated to his low β-glucuronidase activity. Two patients of another family were compound heterozygotes for a C38G and a Y626H allele, and were probably extremely mild MPS VII patients. The low β-glucuronidase activity in another mild MPS VII patient was due to reduced biosynthesis of stable mRNA from one allele, and a W446X mutation on the second. Extremely low β-glucuronidase enzyme activity was also found in the serum of a carrier of a 1801ΔT allele, possibly as a consequence of a dominant-negative effect. A combination of investigations is necessary in order to differentiate between mild disease and pseudodeficiency in individuals with enzyme activities close to the threshold. Received: 31 May 1997 / Accepted: 26 Augsut 1997     

β-Glucuronidase P408S,P415L mutations: evidence that both mutations combine to produce an MPS VII allele in certain Mexican patients

Mucopolysaccharidosis type VII (MPS VII, Sly syndrome) is an autosomal recessively inherited lysosomal storage disease caused by a deficiency in β-glucuronidase. We identified and studied a novel allele containing two C-to-T transitions resulting in P408S and P415L alterations, which is present in homozygous state in one Mexican patient and in heterozygous state in another. None of the previous reports describing mutations in the MPS VII gene include Mexican patients. Expression of either of the mutations individually showed only modest effects on the properties of the enzyme. However, expression of the doubly mutant allele resulted in markedly reduced activity and rapid degradation in an early biosynthetic compartment. Received: 13 December 1995 / Revised: 3 April 1996     

Characterization of the defective beta-glucuronidase activity in canine mucopolysaccharidosis type VII

Canine mucopolysaccharidosis type VII results from deficient activity of lysosomal beta-glucuronidase. Residual enzymatic activity (0.2-1.7% of normal) was detected in tissue homogenates from affected dogs. In contrast, serum and urine from affected animals had up to 15% residual activity. To further characterize the nature of the defective enzyme, hepatic beta-glucuronidase was partially purified from normal and MPS VII dogs for determination of their physical and kinetic properties. About 65% of the total beta-glucuronidase in normal canine liver required detergent for solubilization (i.e., membrane-associated), whereas only 22% of the residual activity in canine MPS VII liver was membrane-associated. Compared to the normal hepatic enzyme, the Km towards 4-methylumbelliferyl-beta-glucuronide was markedly increased in MPS VII dogs (i.e., 0.48 versus greater than 2.5 mmol/l). In contrast, the thermo-, cryo-, and pH stability properties, as well as the pH optimum (approximately 4.6), were essentially unaffected. In addition, the canine MPS VII hepatic residual activity was unresponsive to sulfhydryl reducing reagents and divalent cations, despite the fact that incubation of normal canine beta-glucuronidase with dithiothreitol and magnesium and/or calcium enhanced the activity more than 15-fold.     

Gastrointestinal pathology in a mouse model of mucopolysaccharidosis type IIIA

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by a deficiency in sulphamidase (NS), a lysosomal enzyme required for the degradation of heparan sulphate glycosaminoglycans (gags). The MPS IIIA mouse is a naturally occurring model that accurately reflects the human pathology and disease course. It displays primarily central nervous system pathology accompanied by widespread accumulation of gag in somatic tissues. MPS IIIA mice exhibit greater bodyweight gain than normal littermates and attain a higher mature bodyweight. In this study, gastrointestinal morphology and function was characterised in the IIIA mouse. Stomach and duodenum weight increased in MPS IIIA mice and duodenum length also increased. An increased submucosal thickness was observed in MPS IIIA intestine compared to normal mice and lysosomal storage of gag was observed in this region. Storage was also observed in the lamina propria of the villus tip. All other morphometric measurements including villus height and crypt depth fell within the normal range. The gastric emptying half‐life of solid and liquid meals decreased with age in normal mice whereas the T_½ of solid meals did not alter with age in MPS IIA mice such that they were elevated above normal by 38 weeks of age. Sucrase activity was higher than normal in MPS IIIA at all ages tested. These abnormalities in GI structure and function observed in MPS IIIA may contribute to weight gain in this disorder. J. Cell. Physiol. 219: 259–264, 2009. © 2009 Wiley‐Liss, Inc.     

Hyaluronidase increases the biodistribution of acid alpha-1,4 glucosidase in the muscle of Pompe disease mice: an approach to enhance the efficacy of enzyme replacement therapy

Pompe disease (glycogen storage disease type II) is a glycogen storage disease caused by a deficiency of the lysosomal enzyme, acid maltase/acid alpha-1,4 glucosidase (GAA). Deficiency of the enzyme leads primarily to intra-lysosomal glycogen accumulation, primarily in cardiac and skeletal muscles, due to the inability of converting glycogen into glucose. Enzyme replacement therapy (ERT) has been applied to replace the deficient enzyme and to restore the lost function. However, enhancing the enzyme activity to the muscle following ERT is relatively insufficient. In order to enhance GAA activity into the muscle in Pompe disease, efficacy of hyaluronidase (hyase) was examined in the heart, quadriceps, diaphragm, kidney, and brain of mouse model of Pompe disease. Administration of hyase 3000 U/mouse (intravenous) i.v. or i.p. (intraperitoneal) and 10 min later recombinant human GAA (rhGAA) 20 mg/kg i.v. showed more GAA activity in hyase i.p. injected mice compared to those mice injected with hyase via i.v. Injection of low dose of hyase (3000 U/mouse) or high dose of hyase (10,000 U/mouse) i.p. and 20 min or 60 min later 20 mg/kg rhGAA i.v. increased GAA activity into the heart, diaphragm, kidney, and quadriceps compared to hyase untreated mice. These studies suggest that hyase enhances penetration of enzyme into the tissues including muscle during ERT and therefore hyase pretreatment may be important in treating Pompe disease.