Human Molecular Genetics, 2002, Vol. 11, No. 14 1637-1645
© 2002 Oxford University Press
Muscle as a putative producer of acid 
-glucosidase for glycogenosis type II gene therapy
1Département de Génétique, Développement et Pathologie Moléculaire, Institut Cochin, INSERM U567, Paris, France, 2Université Paris V, Paris, France, 3INSERM U153, Hopital de la Salpétrière, Paris, France, 4Dipartimento di Scienze Biochimiche e Biotecnologie Molecolar, University of Perugia, Italy, 5Généthon III, CNRS URA 1923, Evry, France and 6NIH, Bethesda, MD, USA
Received March 1, 2002; Accepted April 29, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Glycogenosis type II (GSD II) is a lysosomal disorder affecting skeletal and cardiac muscle. In the infantile form of the disease, patients display cardiac impairment, which is fatal before 2 years of life. Patients with juvenile or adult forms can present diaphragm involvement leading to respiratory failure. The enzymatic defect in GSD II results from mutations in the acid
-glucosidase (GAA) gene, which encodes a 76 kDa protein involved in intralysosomal glycogen hydrolysis. We previously reported the use of an adenovirus vector expressing GAA (AdGAA) for the transduction of myoblasts and myotubes cultures from GSD II patients. Transduced cells secreted GAA in the medium, and GAA was internalized by receptor-mediated capture, allowing glycogen hydrolysis in untransduced cells. In this study, using a GSD II mouse model, we evaluated the feasibility of GSD II gene therapy using muscle as a secretary organ. Adenovirus vector encoding AdGAA was injected in the gastrocnemius of neonates. We detected a strong expression of GAA in the injected muscle, secretion into plasma, and uptake by peripheral skeletal muscle and the heart. Moreover, glycogen content was decreased in these tissues. Electron microscopy demonstrated the disappearance of destruction foci, normally present in untreated mice. We thus demonstrate for the first time that muscle can be considered as a safe and easily accessible organ for GSD II gene therapy. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Glycogen storage disease type II (GSD II) is an autosomal recessive disorder caused by mutations in the acid
-glucosidase (GAA) gene (EC 3.2.1.3), which is located at 17q23 in humans (1,2). The physiological role of GAA is to hydrolyze intralysosomal glycogen by cleaving -1,4 and -1,6 glycosidic linkages at acid pH. The glucose released into the cytosol is thus available for the energy needs of cells. In GAA deficiency, glycogen accumulates in the lysosome, leading to tissue impairment, mainly in skeletal muscle, the heart and the diaphragm. Patients with the infantile form of the disease present a generalized hypotonia and a severe cardiomyopathy leading to death before 2 years of life. Juvenile and adult patients develop a progressive muscular weakness, and, in some cases, diaphragm involvement may cause respiratory failure. The heterogeneity of the phenotypes reflects, to a certain extent, the multiplicity of mutations (3–5). No curative treatment is currently available for GSD II patients.
The maturation of GAA results from a multistep processing of precursor forms. The enzyme is synthesized as a 110 kDa precursor form, which is glycosylated and phosphorylated on mannose residues in the endoplasmic reticulum and Golgi apparatus (6). These modifications allow its trafficking through the mannose-6-phosphate (M6P) receptor pathway, which is common to all lysosomal hydrolases (7). The precursor can follow two different routes to the lysosome. It is principally targeted to the lysosome following the intracellular trafficking of M6P receptors but can also be secreted into the extracellular environment. Extracellular GAA can be taken up by ubiquitous cell surface M6P receptors and follow the endocytosis pathway to the lysosome. An M6P-independent pathway has also been described for GAA uptake (8). An intermediate precursor of 95 kDa is formed in the late endosome/lysosome. The 76 and 70 kDa forms of the protein are generated through a last intralysosomal cleavage of precursors by thiol proteinases at acid pH (6). This pattern of GAA trafficking thus provides the basis of a therapeutic strategy for GSD II, since the presence of GAA in the circulation allows distribution to all peripheral organs. In favor of this therapeutic strategy, a partial correction of 20% the normal enzymatic level is sufficient to avoid symptoms: ‘pseudodeficient patients’ with such >20% activity are asymptomatic.
Well-characterized mouse models of GSD II have been generated (9,10). These knockout (GAA-/-) mice are completely GAA-deficient and have a generalized intralysosomal glycogen accumulation (10). They exhibit both early and late features of the disease, with massive storage of glycogen in the heart and a progressive muscular wasting.
One way to supply GAA consists of direct intravenous injection of the recombinant enzyme. Bijvoet et al. (11,12) showed that ‘enzyme replacement’ corrected the enzyme deficit and the glycogen accumulation in the GSD II mouse model. Furthermore, clinical trials are underway to study the feasibility of enzyme replacement for GSD II patients (13,14). This treatment is already established for Gaucher disease (15), and results from clinical trials for Fabry disease are also convincing (16,17). Nevertheless, enzyme replacement is very expensive and constraining for patients. Moreover, in some patients, repetitive injection of the recombinant enzyme induces neutralizing antibodies, the emergence of which compromises the success of treatment (18). As an alternative approach, gene therapy could minimize these inconveniences by providing a continuous production–secretion–recapture of the enzyme. Indeed, transduction of a limited pool of cells should achieve a systemic secretion of the enzyme and its uptake by affected peripheral organs. Two major organs – liver and muscle – have been successfully used for the production and secretion of therapeutic proteins into the circulation. Muscle is an attractive target considering that muscular mass constitutes a large fraction of the body. Successful secretion into the bloodstream has been obtained for factor IX (19), erythropoietin (20) and neurotrophic factor NT-3 (21). The liver is a good producer of lysosomal enzymes (22,23). Amalfitano's team (24) reported the use of an adenovirus vector for the transduction of the liver in GAA-/- mice and subsequent enzymatic correction in peripheral organs.
Here, we investigated the potential of muscle-mediated gene therapy to treat GSD II. Intramuscular injection is a safe and easy way for vector administration. Previously, in our laboratory, an adenovirus vector encoding human GAA (AdGAA) was constructed and characterized in vitro (25). AdGAA transduced myoblasts and myotubes from GSD II patients. The transduced cells overexpressed GAA into the medium and extracellular GAA was taken up by untransduced cells. Moreover, in the corrected untransduced cells, the glycogen accumulation was reduced. Here, we studied the potential of this vector in vivo in muscle of GAA-/- mice. AdGAA was injected at postnatal day 4 in the gastrocnemius. We believe that our results are encouraging, since we obtained an enzymatic correction in peripheral affected organs leading to a remarkable decrease of intralysosomal glycogen content, demonstrating that secretion by skeletal muscle allows a widespread correction of GSD II.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Intramuscular administration of AdGAA leads to GAA overexpression in the injected muscle
In order to test the in vivo capacity of AdGAA to excrete GAA at the therapeutic level, GAA-/- mice received 5x108 pfu of AdGAA in the right gastrocnemius muscle at postnatal day 4. Using AdLacZ as a control, we estimated that approximately 30% of all gastrocnemius muscle fibers were transduced 2 weeks post injection (not shown). The GAA activity was assayed in the injected muscle at 2, 4 and 13 weeks post injection, and reached 138±11, 295±39 and 786±66 nmol/h/mg of protein, respectively (Fig. 1). These activities exceeded those assayed in a normal muscle (C57/B16), which showed 7.5±0.3 nmol/h/mg of protein, whereas in untreated GAA-/- mice, GAA activity was 0.3±0.06 nmol/h/mg of protein.
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To further characterize GAA in the injected muscle, we analyzed the isoenzymes by ionic exchange chromatography on DEAE–cellulose columns (Fig. 2). Both acid (pH 4.0, black points) and neutral (pH 6.5, white points)
-glucosidase activities were assayed. All acid -glucosidase activity from normal muscle resolved in two peaks: one eluted at 0.02 M NaCl and the second eluted within the 0.02–0.3 M NaCl gradient (Fig. 2A). As determined from western blot analysis (not shown), these two peaks correspond to the 70 and 76 kDa mature polypeptides of acid -glucosidase. No activity was detected in GAA-/- mice analyzed under identical conditions (Fig. 2B). In the injected muscle of AdGAA-treated GAA-/- mice, the two mature forms of acid -glucosidase were restored (Fig. 2C). Notably, the activities of these two peaks were a hundred times higher than the normal level. The elution pattern and the relative distribution of GAA activity were identical to those seen in normal muscle. In addition, AdGAA treatment gave rise to a further peak of activity not retained by the column (on the left). This additional peak corresponded to the precursor form (western blot analysis not shown). This form was never detected in normal muscle. GAA activity at neutral pH was analyzed as a control: it was resolved in multiple peaks in a normal muscle, and the pattern in GAA-/- mice, treated or not, had essentially the same profile.
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In Figure 3A, we report the pattern of GAA expression in the injected muscle. It shows the very high quantities of the 70 and 76 kDa mature forms and the intracellular 95 kDa precursor. Moreover, the overexpression of GAA is visualized by the presence of the 110 kDa precursor, which is the secreted form of the enzyme.
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GAA secretion into the plasma
To test if the overexpressed GAA in the injected muscle was secreted into the circulation, the presence of GAA was tested in the plasma. GAA was undetectable in wild-type and in untreated GAA-/- mouse plasma. Western blot analysis showed that the 110 kDa precursor form was present in plasma of AdGAA-injected mice (Fig. 3B).
Before analyzing the distribution of the enzyme in peripheral organs, we questioned whether systemic GAA was due to indirect transduction of other tissues following the intramuscular injection. The liver is the preferential target of a circulating adenovirus vector (26). Thus, if vector particles were able to enter into the bloodstream, some hepatic cells might be transduced, resulting in systemic enzyme secretion. In order to exclude this possibility, we assayed ß-galactosidase activity (X-gal staining) in the livers of animals injected in muscle with the AdLacZ vector; however, no X-gal staining was detected (data not shown). Moreover, in order to test if viral particles were present in this tissue, we assayed DNA from liver by PCR amplification of a fragment overlapping the adenoviral backbone and the human GAA cDNA. As a positive control, we used a liver DNA sample from an animal intravenously injected with AdGAA (Fig. 4). No amplification was obtained in samples of animals injected by the intramuscular route with this vector. This result suggests that there was no or a very limited passage of viral particles via the bloodstream during intramuscular injection.
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GAA activity is restored in affected peripheral muscles after intramuscular injection
As described above, our data show that AdGAA-injected muscle is able to secrete GAA into the bloodstream. The contralateral gastrocnemius muscle served as a control of enzyme uptake at a distance. We found GAA activity in the contralateral muscle at 2 weeks (5.6±2.9 nmol/h/mg of protein), 4 weeks (17±6 nmol/h/mg of protein) and 13 weeks postanjection (4.5±1.3 nmol/h/mg of protein), whereas it was at background levels in GAA-/- untreated muscle (Fig. 5).
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GAA activity in the contralateral muscle of AdGAA-treated GAA-/- mice was also analyzed by ion-exchange chromatography, as described above. The acid
-glucosidase pattern was comparable to that observed for the injected muscle, with three sorting peaks corresponding to the two mature forms and to the precursor form (Fig. 2D). The total activity was observed to be in the normal range as compared with wild-type (C57/B16) mice. The heart, the organ mainly affected in the infantile form of GSD II, was also tested for GAA restoration. Enzyme activity in the heart of treated GAA-/- mice assayed at 2, 4 and 13 weeks post injection corresponded to 0.4±0.2, 0.35±0.2 and 0.52±0.4 nmol/h/mg of proteins, respectively. GAA activity in normal heart reached 4.0 nmol/h/mg of protein, whereas it was at background level in GAA-deficient heart (Fig. 6).
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Intracellular glycogen clearance in mice treated by intramuscular AdGAA administration
To determine the functionality of the muscle-secreted enzyme, intracellular glycogen in skeletal muscle was quantified (see Materials and Methods). In AdGAA-treated animals, glycogen was reduced both in injected muscle with 3±1.2 nmol of glucose released/h/mg of protein and in contralateral muscle with 5±1.8 nmol of glucose released/h/mg of protein, compared with GAA-/- muscle (47±18 nmol of glucose released/h/mg of protein) at 13 weeks (Fig. 7).
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We also assayed intracellular glycogen content by histological analysis. Periodic acid–Schiff (PAS) staining confirmed that glycogen storage was reduced in muscles of AdGAA-treated mice, with a lower staining intensity in both injected and non-injected contralateral muscles versus GAA-/- muscle (Fig. 8). The levels appeared to be near normal (very low staining). In heart, PAS staining also showed a visible decrease in glycogen content in AdGAA-treated mice, with a minor pink staining compared with the intensive coloration observed in control GAA-/- mice.
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Electron-microscopic analysis of muscle after AdGAA treatment
Furthermore, we examined by electron microscopy the structural aspect of the muscular tissue and the intralysosomal trapped material. Numerous glycogen-filled vacuoles (black arrow) were found in the GAA-/- muscle (Fig. 9B, F), and were accompanied by large destruction foci made up of multimembrane vacuoles present in every myofiber (Fig. 9B). These pathological manifestations were absent in normal muscle (Fig. 8A). Our results showed that AdGAA-injected muscle resembled the normal muscle without vacuoles or destruction foci (Fig. 9C). However, the contralateral muscle still contained some glycogen-filled vacuoles, although we found no destruction granules (Fig. 9D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Gene therapy appears to be an attractive treatment for lysosomal disorders, since the transduction of a restricted pool of cells able to secrete the lysosomal enzyme in the circulation might allow widespread enzymatic correction. Lysosomal enzymes, once in the circulation, can be internalized by ubiquitous M6P receptors and then delivered to the lysosome through the endocytic pathway. The therapeutic process is facilitated by the fact that a partial restoration of approximately 10–20% of the normal enzymatic activity is sufficient to prevent the clinical outcome. The liver can be the target of gene transfer, and its use to secrete GAA has already been reported in the mouse model of GSD II (27). Here, we investigated intramuscular delivery of the therapeutic gene, since this is simpler than intravenous injection.
Using AdGAA, we demonstrated that muscle can provide an efficient systemic secretion of GAA. The adenovirus vector was administered during the neonatal period, since the transduction is enhanced by the presence of immature muscle cells, with a higher density of internalization receptors than present on the surface of mature muscle cells (28). Moreover, the immune response against first-generation adenovirus vectors was limited at this stage, hence lengthening the duration of expression, as previously described (29,30). Using these conditions, we obtained a high expression of GAA in the injected muscle 13 weeks post injection. The injected muscle produced the 70 and 76 kDa lysosomal mature forms of the enzyme as in normal muscle, but also a strong expression of the 110 kDa precursor form. This overexpressed precursor form was, in part, secreted into the bloodstream. The form detected in plasma has an apparent molecular weight slightly higher than in the control sample, but this difference can be explained by a very high concentration of protein in plasma samples after concentration, which disturbs migration. Referring to the literature, this secreted precursor should be a 110 kDa form. This precursor was then taken up at a distance, since it was found in untransduced contralateral muscle. The cross-correction was supported by the fact that, in the injected muscle, a third peak appeared (Fig. 2C, on the left) in addition to the two peaks of mature forms present in normal muscle. This additional peak corresponded to the overexpressed precursor form that was also present in the contralateral muscle, as a marker of the uptake precursor from blood circulation that had not yet been processed into mature forms. Moreover, this precursor was driven to the lysosome, as attested by the presence of the two mature forms of GAA in the contalateral muscle. The total activity of the mature enzymes peaked at more than half the normal level at any time of the study in both transduced and untransduced muscles. However, we do not yet have an explanation as to why, in the contralateral muscle, the activity of GAA was greater 4 weeks post injection than 13 weeks post injection, while the activity in the injected muscle increased. This effect could be attributed to a reduced potential of GAA secretion by mature muscle, compared with younger tissue. Therefore, a diminution of secretion would most probably result in an increase in intracellular GAA activity in the injected muscle and a decrease in GAA correction at a distance. Nevertheless, the restored enzymatic activities were sufficient to decrease stored glycogen as quantified by biochemical assays and also visualized by histological analysis, and to improve phenotype appearance in both injected and contralateral muscles. Electron microscopy confirmed notable beneficial effects of the recombinant enzyme in skeletal muscles at 13 weeks. Large vacuoles filled with stored glycogen were ubiquitous in GAA-/- muscle, but were totally absent in the injected muscle of treated animals. However, vacuoles were reduced but still present in the contralateral muscle of treated animals, in which GAA activity was much lower than in the injected muscle. Notably, large lytic foci were found in every cell of GAA-/- muscle, showing evidence of destruction of the tissue, which could reflect the myopathic phenotype associated with the disease. The phenotype of the injected muscle was essentially normal, showing only a slight irregularity of structure. Such effects could be attributed to the injection of the adenovirus vector. However, we can assert that improvement was due to GAA correction and not to adenovirus injection, since tissues from animals having received AdLacZ were similar to those from untreated animals with regard to destruction and numbers of vacuoles (data not shown). In the contralateral muscle, tissue destruction was also prevented by recombinant enzyme. In heart tissue, the enzyme restoration was moderate. Nevertheless, this level appeared sufficient to hydrolyze trapped glycogen as demonstrated by PAS staining.
This study is thus the first report showing the capacity of muscle to secrete a lysosomal enzyme after in vivo transduction with a viral vector. However, another study using the same mouse model of GSD II demonstrated that no GAA secretion was obtained after intramuscular injection (27). This difference could be explained (i) by the fact that the injection of the adenovirus vector was performed in adult mice while the transduction efficiency of the mature muscle cells by this vector is weak (28) and (ii) by a lower capacity of mature tissue to secrete GAA. Our result is consistent with that of Raben et al. (31) showing that conditional muscle-specific expression of GAA can rescue GAA-/- mice, leading to secretion of the enzyme and correction of peripheral organs. An alternative route of trafficking, not clearly characterized but independent of the M6P receptor, might also help the secretion by muscle of this particular lysosomal enzyme (8).
With the aim of exploiting muscle as an enzyme-producing organ, adeno-associated virus (AAV) vector could be considered as a good therapeutic tool. Such a vector has been shown to transduce muscle cells very efficiently, providing long-term expression (32). Its use for GSD II therapy is nevertheless limited by its low cloning capacity. An alternative way to introduce genes in muscle is the electrotransfer of naked DNA. This method appears to be a potentially safe, non-invasive and inexpensive therapeutic approach that has proved its efficiency for the secretion of therapeutic molecules into the bloodstream (33,34). Moreover, gene transfer in muscle has achieved the best long-term secretion in animal models (32,35). Some clinical trials have therefore been started using this organ (36).
In addition to being efficiently delivered by muscle, therapeutic proteins can be modified in order to improve their capacity to cross biological membranes by coupling to a protein transduction domain (37,38). This improvement could be very attractive for GSD II therapy, since it could increase the access of the GAA to all muscles and especially into the heart, where limited entry was observed in our study. This method might also allow the enzyme to cross the blood–brain barrier and to diffuse widely throughout the brain. The possible pathological involvement of glycogen accumulation in the central nervous system (CNS) in the infantile form of GSD II has recently been suggested (39). Thus, such a modification of therapeutic proteins could open new perspectives in the treatment of lysosomal disorders with CNS involvement.
In the meantime, our results demonstrate that GAA can be secreted after efficient transduction of muscle and then recaptured by untransduced tissues, including skeletal muscle and the heart. The level of enzyme correction in these tissues located at a distance from the injected muscle is sufficient to decrease glycogen overload and to avoid the formation of destruction foci. These findings allow reasonable optimism for the prospect of gene therapy for GSD II.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Animals and recombinant adenovirus vector administration
We used an E1–E3 deleted adenovirus vector containing human GAA cDNA under the control of the cytomegalovirus promotor (AdGAA) (25,40). An adenovirus vector encoding the Escherichia coli ß-galactosidase gene (AdLacZ) was used at the same dose, serving as a control of intramuscular injection.
GAA-knockout (GAA-/-) mice were obtained by gene replacement and were fully deficient for GAA activity (10). They received 5x108 pfu of adenovirus vector at postnatal day 4 in the right gastrocnemius. Each group of mice analyzed (treated and control) was made up of three animals. The contralateral (left) gastrocnemius muscle was used to evaluate peripheral uptake of the enzyme in skeletal muscle. Animals injected with the AdLacZ vector were sacrificed 2 weeks post injection. Animals that had received the AdGAA vector were sacrificed at 2, 4 or 13 weeks post injection. For plasma analysis, blood samples were harvested by retro-orbital sampling into heparinized tubes just before sacrifice.
Acid -glucosidase and protein assays
Tissues were homogenized in buffer (Tris 50 mM, NaCl 150 mM, Nonidet P40 0.05% and DTT 1 mM) after freezing in liquid nitrogen. Assays were performed on supernatants obtained after centrifugation of the homogenates (20 000g, 30 min). Protein concentrations were measured according to the Bradford method (Coomassie protein assay reagent kit, PIERCE, 23200).
GAA activity was determined by the cleavage of an artificial substrate, 4-methylumbelliferyl--D-glucoside, at pH 4. Ten microliters of each extract was incubated with 50 µl of the artificial substrate in 96-well microtiter plates for 30 min at 37°C. The reactions were stopped by adding 100 µl of 1.0 M glycine, pH 10. Activities were expressed as nmol/h/mg of protein.
DEAE–cellulose chromatography
Tissue samples were homogenized in 25 mM sodium phosphate buffer, pH 6.0, and then sonicated and incubated in 0.1% (v/v) Nonidet P40 detergent. All procedures were performed at 4°C. The homogenates were centrifuged at 36 000g for 20 min and the supernantants were loaded onto a 1 ml column previously equilibrated with 25 mM sodium phosphate buffer, pH 6.0. After washing with loading buffer, the proteins retained on the column were eluted with 30 ml of 0.02 M NaCl in 25 mM sodium phosphate buffer, pH 6.0, followed by a linear gradient of 0.02–0.3 M NaCl in 60 ml of the same buffer. The column was finally washed with 5 ml of 1.0 M NaCl. The flow rate was 0.5 ml/min. Fractions (1 ml) were collected and assayed for acid and neutral -glucosidase activities using 3 mM 4-methylumbelliferyl--D-glucoside in 0.1 M citric acid/0.2 M Na2PO4 buffer, at pH 4.0 and pH 6.5 respectively. The reactions were stopped by the addition of 2.850 ml of 0.2 M glycine–NaOH buffer, pH 10.4. Fluorescence was measured on a Perkin Elmer LS 3 Fluorimeter (excitation 360 nm, emission 446 nm). Specific activities were expressed as U/mg of protein.
Western blot detection of GAA
For GAA detection in the injected muscle, 60 µg of soluble proteins (obtained as previously described) from an AdGAA-injected mouse, 4 weeks post injection, were electrophoresed in a SDS–8% polyacrylamide gel, then treated as described above for plasma samples.
For GAA detection in plasma, we used samples from normal C57/B16 mice, untreated GAA-/- and AdGAA-treated GAA-/- mice 4 weeks post injection. GAA was first immunoprecipitated using a goat polyclonal antibody raised against human GAA : 100 µl of plasma were mixed with 50 µl of suspension buffer (50 mM Tris, 150 mM NaCl, 0.05% Nonidet P40 and 1.0 mM DTT) and polyclonal goat anti-human GAA antibody, and incubated for 1 h at 4°C. Protein-G–agarose (Immunoprecipitation kit (Protein G) Boheringer Mannheim) was added to the mix and incubated for 4 h at 4°C. The immunocomplex was then rinsed three times in 50 mM Tris, 300 mM NaCl, 0.05% Nonidet P40 and 1.0 mM DTT and resuspended in Laemlli buffer. Samples were electrophoresed in a SDS–8% polyacrylamide gel and electrotransfered to a nitrocellulose membrane. Membranes were blocked in 5% non-fat milk, incubated with a polyclonal goat anti-human GAA antibody and probed with an anti-goat IgG peroxidase-linked antibody (anti-goat IgG, HRP-conjugated, Santa Cruz Biotechnology). Signals were visualized using a chemioluminescence detection system (ECL detection kit, Amersham Pharmacia).
PCR detection of AdGAA vector in liver tissue
We analyzed tissue from mice 4 weeks post injection. DNA was extracted from liver samples by incubation at 37°C in lysis buffer (100 mM Tris–HCl, 5 mM EDTA, 0.2% SDS, 200 mM NaCl and 100 µg/ml proteinase K) and centrifugation for 10 min at 20 000g. Supernatants were isolated, and DNA was precipitated with an equal volume of isopropanol. DNA was then purified by a classical phenol–chloroform method. PCR amplification was performed using conditions previously described (27).
Intracellular glycogen content assay
Intracellular glycogen was quantified by determining the release of glucose from the natural substrate glycogen, calculated as nmol/h/mg of protein with an enzymatic method using hexokinase (kit Gluco-quant, Roche). Tissue homogenates were treated with 33% KOH, boiled for 15 min and then stored on ice for 15 min. To precipitate glycogen, sodium sulfate and absolute ethanol were added and samples were centrifuged (1500g, 10 min, 4°C). The pellet was resuspended in a 300 µl solution of acetic acid. Glycogen concentration was determined by assay of glucose release after digestion of 100 µl of solution with 0.1 U Aspergillus niger amyloglucosidase (Sigma) at 55°C for 30 min.
Optical and electron microscopy for glycogen analysis
For PAS staining, tissues were fixed in 4% formaldehyde and then included in paraffin. Sections of tissues (5 µm) were stained with PAS using standard methods.
For electron-microscopic analysis, tissues were fixed in 2.5% glutaraldehyde for 1 h and then in 0.6% glutaraldehyde in phosphate-buffered saline. Tissues were post-fixed in 1% OsO4 in phosphate-buffered saline and embedded in Epon resin. Ultrathin sections were prepared and stained with uranyl/lead using standard methods.
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ACKNOWLEDGEMENTS |
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We thank E. Kramer and her team for technical assistance with electron microscopy. We thank J.E. Guidotti for scientific advice and critical comments on the manuscript and F. Francis for scrupulous reading of the manuscript. We thank the Vector Core of the University of Nantes supported by the Association Francaise Contre les Myopathies (AFM) for providing the adenovirus vectors. This work was supported by grants from the Association Francophone des Glycogénoses (AFG) and from Vaincre les Maladies Lysosomales (VML).
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FOOTNOTES |
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* To whom correspondence should be addressed at: Département de Génétique, Développement et Pathologie Moléculaire, Institut Cochin, INSERM U567, 24 rue du faubourg Saint-Jacques, 75014 Paris, France. Tel: +33 1 44412401; Email: emt{at}cochin.inserm.fr
Present address: INSERM U505, Institut des Cordeliers, Paris, France.
Present address: Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, Montpellier, France.
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REFERENCES |
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