Differences in the predominance of lysosomal and autophagic pathologies between infants and adults with Pompe disease: implications for therapy

Pompe disease is a lysosomal storage disorder caused by the deficiency of acid alpha-glucosidase, the enzyme that degrades glycogen in the lysosomes. The disease manifests as a fatal cardiomyopathy and skeletal muscle myopathy in infants; in milder late-onset forms skeletal muscle is the major tissue affected. We have previously demonstrated that autophagic inclusions in muscle are prominent in adult patients and the mouse model. In this study we have evaluated the contribution of the autophagic pathology in infants before and 6 months after enzyme replacement therapy. Single muscle fibers, isolated from muscle biopsies, were stained for autophagosomal and lysosomal markers and analyzed by confocal microscopy. In addition, unstained bundles of fixed muscles were analyzed by second harmonic imaging. Unexpectedly, the autophagic component which is so prominent in juvenile and adult patients was negligible in infants; instead, the overwhelming characteristic was the presence of hugely expanded lysosomes. After 6 months on therapy, however, the autophagic buildup becomes visible as if unmasked by the clearance of glycogen. In most fibers, the two pathologies did not seem to coexist. These data point to the possibility of differences in the pathogenesis of Pompe disease in infants and adults.     

Characterization of pre- and post-treatment pathology after enzyme replacement therapy for Pompe disease

In Pompe disease, a genetic deficiency of lysosomal acid alpha-glucosidase, glycogen accumulates abnormally in the lysosomes of skeletal, cardiac and smooth muscle, and contributes to clinically progressive and debilitating muscle weakness. The present study involved 8 infantile-onset Pompe patients, treated weekly with 10 mg/kg of recombinant human acid alpha-glucosidase (rhGAA). Muscle biopsies were obtained at baseline, 12 and 52 weeks post-treatment to establish an indicator of efficacy. Several histologic strategies were employed to characterize changes in pre- and post-treatment samples, including high-resolution light microscopy and digital histomorphometry, electron microscopy, capillary density and fiber type analysis, and confocal microscopy for satellite cell activation analysis. Histomorphometric analysis was performed on muscle samples to assess glycogen depletion in response to enzyme replacement therapy (ERT). The extent of glycogen clearance varied widely among these patient samples, and correlated well with clinical outcome. Low glycogen levels, mild ultrastructural damage, a high proportion of type I fibers, and young age at baseline were all features associated with good histologic response. There was no correlation between capillary density and glycogen clearance, and activated satellite cell levels were shown to be higher in post-treatment biopsies with poor histologic responses. This histopathologic study of infantile Pompe disease provides detailed insight into the cellular progression of the disease and its response to therapy while highlighting a number of methodologies which may be employed to assess regression or progression of the associated pathology. As enzyme replacement therapy becomes more prevalent for the treatment of lysosomal storage diseases, such evaluation of post-treatment pathology will likely become a more common occurrence in the daily practice of pathologists.     

Differential muscular glycogen clearance after enzyme replacement therapy in a mouse model of Pompe disease

Glycogen storage disease in the alpha-glucosidase knockout(6neo(-)/6neo(-)) (GAA KO) mouse, a model of Pompe disease, results in the pathologic accumulation of glycogen primarily within skeletal myocytes and cardiomyocytes. Intravenous administration of recombinant human alpha-glucosidase (rhGAA, Myozyme, aglucosidase alfa) can result in significant glycogen clearance from both cardiomyocytes and skeletal myocytes, however, the degree of clearance varies from one skeletal muscle type to another. We sought to determine what role muscle fiber type predominance played in this variability. To examine this question in the GAA KO mouse model we delivered intravenous doses of 100 mg/kg rhGAA on Day 1, and Day 14, and harvested a variety of fast and slow twitch muscles on Day 28. We measured glycogen clearance, muscle fiber type content and capillary density by light microscopy with computer morphometry. Recombinant human-GAA administration resulted in differential clearance of glycogen in the various muscles examined. Slow twitch-predominant muscles cleared glycogen significantly more efficiently than fast twitch-predominant muscles. There was a strong correlation between capillary density and glycogen clearance (r=0.55), suggesting that at the high doses used in this study the differential glycogen clearance observed between muscles is largely due to differential bioavailability of rhGAA regulated by blood flow.     

Unclassified polysaccharidosis of the heart and skeletal muscle in siblings

We describe a 15-year-old boy and his 19-year-old sister with progressive dilated cardiomyopathy and mild non-progressive proximal lower limb myopathy, secondary to the accumulation of amylopectin-like fibrillar glycogen, (polyglucosan) bodies, in heart and skeletal muscle. Evidence of idiopathic amylopectinosis or polysaccharidosis was demonstrated in heart and skeletal muscle tissue by histology, electron microscopy, biochemical, and genetic analysis. In both siblings the heart muscle stored PAS-positive, proteinase-k resistant and partly diastase resistant granulo-filamentous material, simulating polyglucosan bodies. Glycogen branching enzyme activity, and phosphofructokinase enzyme activity, measured in skeletal muscle tissue and explanted heart tissue were all within the normal limits, however glycogen content was elevated. Furthermore, GBE1, PRKAG2, desmin, alphabeta-crystallin, ZASP, myotilin, and LAMP-2 gene sequencing revealed no mutation, excluding e.g. glycogen storage disease type 4 and desmin-related myofibrillar cardiomyopathies. In both patients the diagnosis of an idiopathic polysaccharidosis with progressive dilated cardiomyopathy was made, requiring heart transplantation at age 13 and 14, respectively. Both patients belong to an autosomal recessive group of biochemically and genetically unclassified severe vacuolar glycogen storage disease of the heart and skeletal muscle. Up to now unidentified glycogen synthesis or glycogen degradation pathways are supposed to contribute to this idiopathic glycogen storage disease.     

Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease

The role of autophagy, a catabolic lysosome-dependent pathway, has recently been recognized in a variety of disorders, including Pompe disease, the genetic deficiency of the glycogen-degrading lysosomal enzyme acid-alpha glucosidase. Accumulation of lysosomal glycogen, presumably transported from the cytoplasm by the autophagic pathway, occurs in multiple tissues, but pathology is most severe in skeletal and cardiac muscle. Skeletal muscle pathology also involves massive autophagic buildup in the core of myofibers. To determine if glycogen reaches the lysosome via autophagy and to ascertain whether autophagic buildup in Pompe disease is a consequence of induction of autophagy and/or reduced turnover due to defective fusion with lysosomes, we generated muscle-specific autophagy-deficient Pompe mice. We have demonstrated that autophagy is not required for glycogen transport to lysosomes in skeletal muscle. We have also found that Pompe disease involves induction of autophagy but manifests as a functional deficiency of autophagy because of impaired autophagosomal-lysosomal fusion. As a result, autophagic substrates, including potentially toxic aggregate-prone ubiquitinated proteins, accumulate in Pompe myofibers and may cause profound muscle damage.     

Modulation of glycogen synthesis by RNA interference: towards a new therapeutic approach for glycogenosis type II

Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by defects in the acid alpha-glucosidase gene, which leads to lysosomal glycogen accumulation and enlargement of the lysosomes mainly in cardiac and muscle tissues, resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severely affected patients. Enzyme replacement therapy has already proven to be beneficial in this disease, but correction of pathology in skeletal muscle still remains a challenge. As substrate deprivation was successfully used to improve the phenotype in other lysosomal storage disorders, we explore here a novel therapeutic approach for GSDII based on a modulation of muscle glycogen synthesis. Short hairpin ribonucleic acids (shRNAs) targeted to the two major enzymes involved in glycogen synthesis, i.e. glycogenin (shGYG) and glycogen synthase (shGYS), were selected. C2C12 cells and primary myoblasts from GSDII mice were stably transduced with lentiviral vectors expressing both the shRNAs and the enhanced green fluorescent protein (EGFP) reporter gene. Efficient and specific inhibition of GYG and GYS was associated not only with a decrease in cytoplasmic and lysosomal glycogen accumulation in transduced cells, but also with a strong reduction in the lysosomal size, as demonstrated by confocal microscopy analysis. A single intramuscular injection of recombinant AAV-1 (adeno-associated virus-1) vectors expressing shGYS into newborn GSDII mice led to a significant reduction in glycogen accumulation, demonstrating the in vivo therapeutic efficiency. These data offer new perspectives for the treatment of GSDII and could be relevant to other muscle glycogenoses.     

Murine muscle cell models for Pompe disease and their use in studying therapeutic approaches

Lysosomes filled with glycogen are a major pathologic feature of Pompe disease, a fatal myopathy and cardiomyopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme, acid α-glucosidase (GAA). To facilitate studies germane to this genetic disorder, we developed two in vitro Pompe models: myotubes derived from cultured primary myoblasts isolated from Pompe (GAA KO) mice, and myotubes derived from primary myoblasts of the same genotype that had been transduced with cyclin-dependent kinase 4 (CDK4). This latter model is endowed with extended proliferative capacity. Both models showed extremely large alkalinized, glycogen-filled lysosomes as well as impaired trafficking to lysosomes. Although both Pompe tissue culture models were derived from fast muscles and were fast myosin positive, they strongly resemble slow fibers in terms of their pathologic phenotype and their response to therapy with recombinant human GAA (rhGAA). Autophagic buildup, a hallmark of Pompe disease in fast muscle fibers, was absent, but basal autophagy was functional. To evaluate substrate deprivation as a strategy to prevent the accumulation of lysosomal glycogen, we knocked down Atg7, a gene essential for autophagosome formation, via siRNA, but we observed no effect on the extent of glycogen accumulation, thus confirming our recent observation in autophagy-deficient Pompe mice [N. Raben, V. Hill, L. Shea, S. Takikita, R. Baum, N. Mizushima, E. Ralston, P. Plotz, Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease, Hum. Mol. Genet. 17 (2008) 3897–3908] that macroautophagy is not the major route of glycogen transport to lysosomes. The in vitro Pompe models should be useful in addressing fundamental questions regarding the pathway of glycogen to the lysosomes and testing panels of small molecules that could affect glycogen biosynthesis or speed delivery of the replacement enzyme to affected lysosomes.     

Enzyme replacement therapy in alpha-mannosidosis guinea-pigs

alpha-Mannosidosis is a lysosomal storage disorder caused by deficient activity of lysosomal alpha-mannosidase and is characterised by massive accumulation of mannose-containing oligosaccharides in affected individuals. Patients develop behaviour and learning difficulties, skeletal abnormalities, immune deficiency and hearing impairment. Disease in alpha-mannosidosis guinea-pigs resembles the clinical, histopathological, biochemical and molecular features of the human disease. We have used the guinea-pig model to investigate efficacy of enzyme replacement therapy as a treatment for alpha-mannosidosis. Intravenous recombinant human lysosomal alpha-mannosidase, administered at a dose of 1mg/kg, was cleared from circulation with a half-life of 53 h, with significant enzyme activity (1.4x normal levels) detected in circulation one week post-injection. alpha-Mannosidase administered to alpha-mannosidosis guinea-pigs at 1mg/kg (onset at birth or approximately 30 days) and 10mg/kg (at birth) was distributed widely amongst tissues, including to capillary depleted brain. By monitoring with tandem mass spectrometry, enzyme replacement therapy was found to be effective in reducing stored substrates in peripheral tissues at both dose rates, and in brain by up to 39% at the 10mg/kg dose, compared with untreated alpha-mannosidosis controls. Reductions of up to 60% of urinary mannose containing oligosaccharides were also observed. No histological improvements were seen in the brain at either dose, however marked decreases in lysosomal vacuolation in liver, kidney, spleen and endocrine pancreas, as well as a significant reduction in trigeminal ganglion neurons were observed. Multiple injections of 1mg/kg recombinant enzyme in alpha-mannosidosis guinea-pigs induced a very rapid humoral immune response precluding long-term intravenous treatment.     

Adult-onset acid maltase deficiency. Morphologic and biochemical abnormalities reproduced in in cultured muscle.

We established muscle-tissue cultures from biopsy of a patient with adult-onset acid maltase deficiency. Morphologically and biochemically, the newly grown fibers of the cultured muscle showed the same abnormalities as those of the biopsied muscle. Light microscopy showed multiple vacuoles filled with acid-phosphatase-positive material; on ultrastructural examination there was abnormal accumulation of glycogen in membrane-bound sacs (secondary lysosomes), some of which also contained dark membranous of homogeneous material. Acid maltase (pH 4.0), a lysosomal enzyme, was undetectable in either cultured or biopsied muscle by maltose hydrolysis, whereas acid phosphatase, also a lysosomal enzyme, was increased in both sources of muscle cells. Cultured muscle fibers demonstrate the same morphologic and biochemical abnormalities characteristic of biopsied muscle, supporting the concept of a biochemically distinct primary myopathy in man.     

Physiological role of skeletal muscle glycogen in starved mice

To study the physiological role of skeletal muscle glycogen in starved animals, effects of starvation on glycogen and glycogen phosphorylase (EC 2.4.1.1.) activity were studied in muscle fibers (morphologic study) and in whole muscles (biochemical study) of the rectus femoris muscle of mouse. Glycogen content in the liver of the starved animals was also measured. PAS reaction, strong in muscle fibers of fed animals, became weak predominantly in type IIB fibers after 2 days and almost disappeared after 4 days of starvation. Glycogen particles, numerous in the sarcoplasm between myofibrils of muscle fibers, decreased markedly predominantly in type IIB fibers after 2 days and almost disappeared after 4 days. Phosphorylase a activity, undetected in fibers of fed mice, appeared weak in type IIB fibers and very weak in type IIA fibers after 2 days and became moderate in type IIB fibers and weak in type IIA fibers after 4 days. Muscle glycogen content did not differ by 16 hours from the values of corresponding fed animals. However, liver glycogen content had already decreased after 8 hours and markedly so after 12 hours. The results support our hypothesis-"skeletal muscle glycogen is used for maintaining the blood glucose level in starved mice" (Hirose et al.: Anat. Rec., 216:133-138, 1986)-and show that type IIB fibers play a main role in maintaining the glucose level and that muscle glycogen is utilized after depletion of liver glycogen.     

Defect in degradation of glycogenin-exposed residual glycogen in lysosomes is the fundamental pathomechanism of Pompe disease

Pompe disease is a lysosomal storage disorder that preferentially affects muscles, and it is caused by GAA mutation coding acid alpha-glucosidase in lysosome and glycophagy deficiency. While the initial pathology of Pompe disease is glycogen accumulation in lysosomes, the special role of the lysosomal pathway in glycogen degradation is not fully understood. Hence, we investigated the characteristics of accumulated glycogen and the mechanism underlying glycophagy disturbance in Pompe disease. Skeletal muscle specimens were obtained from the affected sites of patients and mouse models with Pompe disease. Histological analysis, immunoblot analysis, immunofluorescence assay, and lysosome isolation were utilized to analyze the characteristics of accumulated glycogen. Cell culture, lentiviral infection, and the CRISPR/Cas9 approach were utilized to investigate the regulation of glycophagy accumulation. We demonstrated residual glycogen, which was distinguishable from mature glycogen by exposed glycogenin and more α-amylase resistance, accumulated in the skeletal muscle of Pompe disease. Lysosome isolation revealed glycogen-free glycogenin in wild type mouse lysosomes and variously sized glycogenin in Gaa^−/− mouse lysosomes. Our study identified that a defect in the degradation of glycogenin-exposed residual glycogen in lysosomes was the fundamental pathological mechanism of Pompe disease. Meanwhile, glycogenin-exposed residual glycogen was absent in other glycogen storage diseases caused by cytoplasmic glycogenolysis deficiencies. In vitro, the generation of residual glycogen resulted from cytoplasmic glycogenolysis. Notably, the inhibition of glycogen phosphorylase led to a reduction in glycogenin-exposed residual glycogen and glycophagy accumulations in cellular models of Pompe disease. Therefore, the lysosomal hydrolysis pathway played a crucial role in the degradation of residual glycogen into glycogenin, which took place in tandem with cytoplasmic glycogenolysis. These findings may offer a novel substrate reduction therapeutic strategy for Pompe disease. © 2024 The Pathological Society of Great Britain and Ireland.     

Synthesis and In Situ Localization of Lysosomal α-Glucosidase in Muscle of an Unusual Variant of Glycogen Storage Disease Type II

The lysosomal α-glucosidase activity is reduced to 10% to 25% of the average control value in most late-onset cases of glycogen storage disease type II (GSDII). Some adult patients, however, have been identified with an exceptionally low (<5%) residual enzyme activity. We have investigated one such unusual variant. The rate of α-glucosidase synthesis appeared normal but the residual enzyme activity was only approximately 3% in cultured fibroblasts, cultured muscle cells, and muscle tissue of the patient. It appeared that fully matured enzyme molecules were more abundantly present in muscle tissue than in cultured cells. The acid phosphatase activity of affected muscle fibers was enhanced due to an increased number of lysosomes. Lysosomes were particularly abundant in vacuolated areas and they contained, as judged by immunoelectron microscopy, even more α-glucosidase molecules than usual. An excessive amount of enzyme molecules were also observed in the endoplasmic reticulum, the site of lysosomal enzyme synthesis, and the cisternae were dilated. These observations suggest that the lysosomal system is stimulated in response to intralysosomal glycogen storage and onset of cellular injury. We hypothesize that the onset of gross pathologic abnormalities is delayed in this particular case of adult GSDII by an increased synthesis of lysosomal α-glucosidase, and as a consequence, an increased residual activity in storage-prone muscle fibers.     

Generalized glycogen storage and cardiomegaly in a knockout mouse model of Pompe disease

Glycogen storage disease type II (GSDII; Pompe disease), caused by inherited deficiency of acid alpha-glucosidase, is a lysosomal disorder affecting heart and skeletal muscles. A mouse model of this disease was obtained by targeted disruption of the murine acid alpha-glucosidase gene (Gaa) in embryonic stem cells. Homozygous knockout mice (Gaa -/-) lack Gaa mRNA and have a virtually complete acid alpha-glucosidase deficiency. Glycogen-containing lysosomes are detected soon after birth in liver, heart and skeletal muscle cells. By 13 weeks of age, large focal deposits of glycogen have formed. Vacuolar spaces stain positive for acid phosphatase as a sign of lysosomal pathology. Both male and female knockout mice are fertile and can be intercrossed to produce progeny. The first born knockout mice are at present 9 months old. Overt clinical symptoms are still absent, but the heart is typically enlarged and the electrocardiogram is abnormal. The mouse model will help greatly to understand the pathogenic mechanism of GSDII and is a valuable instrument to explore the efficacy of different therapeutic interventions.      

Glycogen debranching enzyme is associated with rat skeletal muscle sarcoplasmic reticulum

AIMS: Gel electrophoresis revealed a band of molecular weight approximately 160 000 Da associated with the skeletal muscle sarcoplasmic reticulum (SR) vesicle preparations. This investigation sought to examine glycogen debranching enzyme associated with skeletal muscle SR. METHODS: Sarcoplasmic reticulum samples were also taken from muscle whose glycogen content had been reduced either via stimulation of the sciatic nerve or alpha-amylase treatment of muscle homogenates. RESULTS: The stimulation protocol reduced whole muscle glycogen by 86% (7.4 +/- 0.4 vs. 1.0 +/- 0.3 microg mg(-1) wet mass, P < or = 0.05). Glycogen associated with the SR was reduced by 82% in the stimulation protocol (533 +/- 82 vs. 96 +/- 7 microg mg(-1) protein) and by 94% in alpha-amylase treatment (493 +/- 11 vs. 29 +/- 2 microg mg(-1) protein), respectively. Gel electrophoresis and Western blots revealed that the content of glycogen debranching enzyme was reduced by approximately 53% as a result of muscle stimulation and by approximately 46% in alpha-amylase treatment (P < or = 0.05). In addition, glycogen debranching enzyme activity was reduced by 61% in stimulated samples compared with control (20.3 +/- 1.0 vs. 8.0 +/- 1.2 nmol mg(-1) min(-1), respectively), a value consistent with reductions observed from gel electrophoresis and Western blots. CONCLUSION: These results confirm that similar to glycogen phosphorylase, glycogen debranching enzyme is associated with the skeletal muscle SR and is dissociated under exercise conditions.     

Human acid alpha-glucosidase from rabbit milk has therapeutic effect in mice with glycogen storage disease type II.

Pompe's disease or glycogen storage disease type II (GSDII) belongs to the family of inherited lysosomal storage diseases. The underlying deficiency of acid alpha-glucosidase leads in different degrees of severity to glycogen storage in heart, skeletal and smooth muscle. There is currently no treatment for this fatal disease, but the applicability of enzyme replacement therapy is under investigation. For this purpose, recombinant human acid alpha-glucosidase has been produced on an industrial scale in the milk of transgenic rabbits. In this paper we demonstrate the therapeutic effect of this enzyme in our knockout mouse model of GSDII. Full correction of acid alpha-glucosidase deficiency was obtained in all tissues except brain after a single dose of i.v. enzyme administration. Weekly enzyme infusions over a period of 6 months resulted in degradation of lysosomal glycogen in heart, skeletal and smooth muscle. The tissue morphology improved substantially despite the advanced state of disease at the start of treatment. The results have led to the start of a Phase II clinical trial of enzyme replacement therapy in patients.     

Response of mouse liver glycogen cycle enzymes to endotoxin treatment.

The present study was undertaken to characterize endotoxin-induced changes in carbohydrate metabolism and more specifically, to determine the contribution of glycogenolysis to the loss of liver glycogen. Female ICR mice, fasted overnight, were injected with a median lethal dose (LD50, 9 mg/kg) of endotoxin extracted from Salmonella typhimurium strain SR-11. Glycogen synthase and glycogen phosphorylase activities were measured at 0.5 and 6 h after treatment. Endotoxin treatment did not alter total glycogen synthase activity, but the amount of enzyme present in the active form was significantly lower in endotoxic mice. There was no significant increase in glycogen phosphorylase activity in endotoxin-treated mice. Glycogen phosphorylase was activated to the same extent in control and endotoxic mice by decapitation or intravenous epinephrine (25 or 1 mug/kg). The results of this study indicate no significant increase in glycogen phosphorylase activity in endotoxic mice, contraindicating enhanced glycogenolysis as a mechanism for depletion of carbohydrate following endotoxin injection. Altered activation of glycogen synthase, however, may contribute to the loss of glycogen during endotoxemia.     

Changes in glycogen and glycosaminoglycan levels in hepatocytes of iduronate-2-sulfatase knockout mice before and after recombinant iduronate-2-sulfatase supplementation

Purpose

Mucopolysaccharidosis II (MPS II) is a lysosomal storage disorder caused by a deficiency of iduronate-2 sulfatase (IdS), which is involved in the degradation of glycosaminoglycan (GAG). In this study, the frequency of fasting hypoglycemia in patients with MPS II was investigated and changes in accumulation of glycogen and GAG in the hepatocytes of IdS-knockout (KO) mice were evaluated before and after recombinant IdS enzyme replacement therapy (ERT).

Materials and Methods

Plasma glucose levels were evaluated after an 8-hour fast in 50 patients with MPS II. The IdS-KO mice were divided into three groups (group 2; saline, group 3; 0.15 mg/kg of IdS, and group 4; 0.5 mg/kg of IdS); wild-type mice were included as controls (group 1). ERT was initiated intravenously at four weeks of age, and continued every week until 20 weeks of age.

Results

The mean glucose level after an 8-hour fast was 94.1 ± 23.7 mg/dL in the patients with MPS II. Two (4%) out of 50 patients had fasting hypoglycemia. For the mice, GAG in the lysosomes nearly disappeared and glycogen particles in the cytoplasm were restored to the normal range in group 4.

Conclusion

Glucose metabolism in patients with MPS II appeared to function well despite hepatocytic GAG accumulation and hypothetical glycogen depletion. A higher dose of IdS infusion in MPS II mice led to disappearance of lysosomal GAG and restoration of glycogen to the cytoplasm of hepatocytes.     

Recombinant human acid alpha-glucosidase: high level production in mouse milk, biochemical characteristics, correction of enzyme deficiency in GSDII KO mice

Glycogen storage disease type II (GSDII) is caused by lysosomal acid alpha-glucosidase deficiency. Patients have a rapidly fatal or slowly progressive impairment of muscle function. Enzyme replacement therapy is under investigation. For large-scale, cost-effective production of recombinant human acid alpha-glucosidase in the milk of transgenic animals, we have fused the human acid alpha-glucosidase gene to 6.3 kb of the bovine alphaS1-casein gene promoter and have tested the performance of this transgene in mice. The highest production level reached was 2 mg/ml. The major fraction of the purified recombinant enzyme has a molecular mass of 110 kDa and resembles the natural acid alpha-glucosidase precursor from human urine and the recombinant precursor secreted by CHO cells, with respect to pH optimum, Km, Vmax, N-terminal amino acid sequence and glycosylation pattern. The therapeutic potential of the recombinant enzyme produced in milk is demonstrated in vitro and in vivo. The precursor is taken up in a mannose 6-phosphate receptor-dependent manner by cultured fibroblasts, is converted to mature enzyme of 76 kDa and depletes the glycogen deposit in fibroblasts of patients. When injected intravenously, the milk enzyme corrects the acid alpha-glucosidase deficiency in heart and skeletal muscle of GSDII knockout mice.      

Pathological features of glycogen storage disease type II highlighted in the knockout mouse model.

Glycogen storage disease type II (GSDII; Pompe's disease) is an autosomal recessive disease caused by lysosomal alpha-glucosidase deficiency. Skeletal muscle weakness is the most conspicuous clinical symptom of patients suffering from GSDII and skeletal muscle also is prominently involved in the knockout mouse model of this disease. Thus far, however, little detailed information has been published on the pathological changes in other mouse tissues. This paper aims to provide these data and gives a record of the clinical course of the mouse model over a 2-year period. Four-month-old affected mice perform worse in a running wheel than their unaffected littermates, but do not yet display other clear signs of disease. The lysosomal glycogen storage, already evident at birth, becomes more severe in time, leading to muscle wasting by 9-10 months of age and then limb girdle weakness and kyphosis. The disease does not markedly shorten the animal's life span despite the serious tissue pathology, which is not limited to heart and skeletal muscle, but is also seen in the smooth muscle of blood vessels and of the respiratory, digestive, and urogenital tracts. In addition, the mice have lysosomal glycogen storage in the liver, kidney, spleen, and salivary gland; in Schwann cells of the peripheral nerves, and in a subset of neurons in the central nervous system. By pathological criteria, the knockout mouse model parallels the human infantile form of GSDII and is attractive for studying the possible reversal of tissue pathology and symptomatology under different therapeutic regimes.