Critical Review. New Insights into Therapeutic Options for Pompe Disease. Emmanuel Richard 1,2, Gaëlle Douillard-Guilloux 3 and Catherine Caillaud 3

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1 IUBMB Life, 63(11): , November 2011 Critical Review New Insights into Therapeutic Options for Pompe Disease Emmanuel Richard 1,2, Gaëlle Douillard-Guilloux 3 and Catherine Caillaud 3 1 Université de Bordeaux, Biothérapies des Maladies Génétiques et Cancers, U1035, F Bordeaux, France 2 INSERM, Biothérapies des Maladies Génétiques et Cancers, U1035, F Bordeaux, France 3 Université Paris Descartes, Paris, France; INSERM U845, Paris, France Summary Glycogen storage disease type II or Pompe disease (GSD II, MIM ) is a rare inherited metabolic myopathy caused by a deficiency of lysosomal acid a-glucosidase or acid maltase (GAA; EC ), resulting in a massive lysosomal glycogen accumulation in cardiac and skeletal muscles. Affected individuals exhibit either severe hypotonia associated with hypertrophic cardiomyopathy (infantile forms) or progressive muscle weakness (late-onset forms). Even if enzyme replacement therapy has recently become a standard treatment, it suffers from several limitations. This review will present the main results of enzyme replacement therapy and the recent findings concerning alternative treatments for Pompe disease, such as gene therapy, enzyme enhancement therapy, and substrate reduction therapy. Ó 2011 IUBMB IUBMB Life, 63(11): , 2011 Keywords lysosomal storage disease; glycogenosis type II; Pompe disease; glycogen metabolism; metabolic myopathy; substrate reduction therapy; pharmacological chaperone; gene therapy. POMPE DISEASE IN PATIENTS AND KNOCK-OUT MICE Pompe disease, also known as glycogen storage disease type II (GSD II) or acid maltase deficiency (MIM ), is an autosomal recessive neuromuscular disorder caused by mutations in the gene that encodes the lysosomal hydrolase acid a-glucosidase (GAA; EC ) (1). The deficiency of this enzyme results in lysosomal glycogen accumulation in multiple Received 6 April 2011; accepted 31 May 2011 Address correspondence to: Emmanuel Richard, Université Victor Segalen Bordeaux 2, INSERM U1035, 146 rue Léo Saignat, Bordeaux Cedex, France. Emmanuel.Richard@u-bordeaux2.fr or Catherine Caillaud, INSERM U845, 156 rue de Vaugirard, Paris, France. Tel: , Fax: catherine.caillaud@inserm.fr tissues, responsible for cellular dysfunction, particularly in skeletal and cardiac muscle. Epidemiological studies have estimated the combined incidence of all clinical forms around 1 for 40,000 live births. GSD II encompasses a broad spectrum of phenotypes that range from the severe infantile-onset form to the more slowly progressive late-onset form (2). The infantile form is characterized by generalized hypotonia, muscle weakness, and hypertrophic cardiomyopathy leading to death during the first year of life due to cardiac and/or respiratory failure. Late onset forms present with progressive muscle weakness mainly involving proximal muscle and diaphragm and resulting in premature death from respiratory failure. The different clinical forms of GSD II are due to a large variety of mutations on the GAA gene. Among them, some common mutations have been reported: the mutations c _ del (D exon 18) and c.525delt are usually found in infantile forms, whereas the mutation c t[g in intron 1 is generally associated with late-onset forms of the disease. Nutrition (high-protein and low-carbohydrate diet) and exercise therapy have been used in patients with late-onset GSD II in order to slow muscle deterioration, but this approach is only palliative. Specific therapies have been developed in the last years and they will be discussed here. Different GSD II murine models have been generated in order to facilitate pathophysiological studies as well as development of therapeutic strategies. These models were obtained by homologous recombination in ES cells, and they specifically targeted exon 6, 13, or 14 (3 5). Knock-out (KO) mice exhibited generalized and progressive glycogen accumulation in liver, heart, and skeletal muscle. However, clinical expression of the disease was highly variable among the models. Features of both adult and infantile phenotype (muscle weakness, cardiomegaly) were observed in 6 neo /6 neo mice, while mice with disrupted exon 13 remain phenotypically normal. An immunodeficient GAA-KO murine model (GAA-KO/SCID) was developed to avoid humoral response against recombinant enzyme (5). This model has allowed long-term studies of GAA enzymatic restoration under ERT and has widely been used in gene therapy experiments. ISSN print/issn online DOI: /iub.529

2 980 RICHARD ET AL. RECENT FINDINGS ON PATHOGENESIS OF THE MUSCULAR DAMAGE GAA deficiency leads to progressive accumulation of lysosomal glycogen mainly in cardiac and skeletal muscles. The underlying mechanism of muscle weakness remains largely unknown and is probably multifactorial. Based on ultrastructural modifications of myocytes in Pompe patients, several stages of disease progression have been described (6). Initially, muscle cells contain small, glycogen-filled lysosomes that progressively increased in size and number. Membranes of swollen lysosomes start to disrupt leading to glycogen leakage into the cytoplasm. Alterations of the mitochondrial architecture as well as disintegration of muscular ultrastructure are frequently observed in Pompe patients (6). In the later stages, myofibers are swollen due to uptake of water which leads to dilution of glycogen (6, 7). The disruption of contractile units by numerous noncontractile inclusions, such as swollen lysosomes and autophagosomes may account for an inadequate myofibrillar transmission force (7). Furthermore, X-ray diffraction analysis revealed alterations of the initial actin-myosin interactions that can contribute to skeletal muscle weakness in GAA-KO mice (8). Autophagy is a catabolic lysosome-dependent degradative pathway of intracellular components that was pointed out to play a role in the pathogenesis of Pompe disease (9). Accumulation of autophagic vacuoles could indicate either an upregulation of autophagy or an incomplete autophagic flux due to defects in autophagosome/lysosome fusion (10). GAA-KO mice show an extensive autophagic buildup significantly contributing to muscle damages in glycolytic fast, but not in oxidative slow, muscle fibers. These inclusions disturb the endosomal/autophagosomal pathway and prevent the efficient delivery of recombinant GAA (rhgaa) into lysosomes of ERT-treated mice. Studies using muscle-specific inactivation of several genes required for macroautophagy (Atg5) in GAA-KO mice revealed that both deficient and excessive autophagy may coexist (11). Autophagy is upregulated in both type I and type II myofibers of GAA-KO mice as shown by studying genes involved in the formation and maturation of autophagosomes. Inefficient disposal of autophagic cargo and subsequent accumulation of ubiquitin-positive bodies were found in fast fibers only and they may be involved in muscle injury. Recently, the contribution of the autophagic pathology has been studied in children with Pompe disease (12). Unexpectedly, this mechanism mainly involved in the pathogenesis of late-onset forms seems negligible in early onset patients, underlining a possible pathophysiological difference between both forms of GSD II. ENZYME REPLACEMENT THERAPY ERT in Mice and Human Enzyme replacement therapy (ERT) is based on the concept that recombinant lysosomal enzymes can be internalized by cells through the mannose-6-phosphate receptor (MPR) pathway (commonly used by newly synthesized and secreted enzymes) and then delivered to lysosomes where they are further processed to replace the function of deficient hydrolases (13) (Fig. 1). In Pompe disease, large-scale production of recombinant human acid a-glucosidase (rhgaa) was obtained in Chinese hamster ovary (CHO) cells and in transgenic rabbit milk (14 16). Administration of rhgaa of both origins led to an increase of GAA activity in muscle, heart, and liver. However, these studies demonstrated an efficient glycogen clearance in cardiac muscle and liver while a modest effect was observed in skeletal muscle (17, 18). Clinical studies based on the administration of rhgaa in classical infantile Pompe patients showed a prominent effect on cardiac hypertrophy, motor skill improvement as well as substantial life-span increase (19 22). Administration of recombinant enzyme in late-onset patients results in a mild improvement of motor and respiratory functions, but ERT efficacy in these patients needs to be evaluated at long-term (23, 24). These studies demonstrated that the outcome is more robust if treatment starts early in the course of the disease. This emphasizes the necessity of an early diagnosis possibly based on systematic screening programs (25, 26), even if presymptomatic diagnosis of adult forms of GSD II has to be discussed (27). Limitations to ERT Even if CHO-derived rhgaa (alglucosidase alfa, Myozyme 1 /Lumizyme 1, Genzyme, Cambridge, MA) was approved in the USA, Europe, and Canada in 2006 and subsequently in numerous countries, becoming the standard treatment for Pompe disease, some drawbacks appeared. (1) A major limitation to ERT is the requirement for intravenous (i.v.) injection of a particularly high dose of recombinant enzyme (20 mg/kg, every 2 weeks), compared with other ERT for lysosomal storage diseases (13). Furthermore, the need for life-long repeated infusions of the recombinant enzyme makes ERT a very expensive treatment. (2) The rhgaa is poorly targeted to muscle and mainly trapped in liver leading to 80% loss of the administered enzyme (16). (3) An ineffective response of type II skeletal muscle fibers to ERT was clearly described in GAA-KO mice (16), due to the dysregulation of the autophagic pathway in glycolytic type II myofibers leading to rhgaa retention in autophagosomes and mistargeting to lysosomes (28, 29). In humans, the situation remains unclear but both type I and type IIA muscle fibers seem able to respond to ERT in infantile forms of Pompe disease (30). (4) Repeated infusions of high amount of exogenous enzyme often lead to the induction of an immune response, especially in cross-reactive immunological material (CRIM)-negative patients with infantile forms (31). (5) The potential contribution of a neural deficit resulting from glycogen storage into the central nervous system (CNS) has been highlighted in GAA-deficient mice and patients. Although glycogen is normally absent from neurons, several reports demonstrated glycogen accumulation in spinal cord, sensory ganglia, and brain leading to degeneration of axons (32, 33). Progressive phrenic nerve injury could contribute to respiratory insufficiency

3 NEW INSIGHTS INTO THERAPEUTIC OPTIONS FOR POMPE DISEASE 981 Figure 1. Possible levels of therapeutic intervention in Pompe disease. The figure shows the different cellular steps of acid maltase synthesis and subsequent glycogen degradation. ER: endoplasmic reticulum, MPR: mannose-6-phosphate receptor, and mrna: messenger RNA. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] in late-onset GSD II patients. Contribution of neuronal glycogen storage to the pathology is not completely elucidated, but it is clear that delivery of the therapeutic protein to the CNS is unlikely due to the blood-brain barrier impermeability. These limitations to ERT have encouraged researchers to develop alternative therapeutic strategies for Pompe disease. GENE THERAPY Gene therapy is an attractive option for Pompe disease because it may allow a long-term correction of GAA deficiency. In vitro studies using retro/lentiviral and adenoviral vectors showed an efficient expression of hgaa cdna in human and murine myoblasts as well as secretion of GAA and reuptake by neighboring cells through MPR endocytosis (34 36) (Fig. 1). In vivo studies were then extensively explored in animal models of Pompe disease, especially in GAA-KO mice. Muscle and Liver Targeted Gene Therapy Raben et al. (37) developed transgenic GAA-KO mice with inducible GAA expression either into liver or into cardiac/skeletal muscle. They demonstrated that while both tissues can produce recombinant GAA, a negligible level of secretion was obtained from muscle, which mediates only a modest metabolic cross-correction. On the contrary, a high hepatic production of the enzyme led to muscle glycogen clearance, indicating that liver could be the best organ for a secretion of GAA. Multiple adenoviral (Ad) and adenoassociated (AAV) vectors of various serotypes have demonstrated their efficacy to transduce muscle, which can be considered as the natural target in GAA- KO mice. Direct intramuscular administration of Ad or AAV2/1 and AAV2/6 vectors containing the muscle-specific creatine kinase (MCK) promoter/enhancer resulted in long-term correction of glycogen content in injected muscle, but failed to provide metabolic cross-correction into distant noninjected muscles (38 40). Direct injections of GAA-expressing AAV2/1 vector in the diaphragm of GAA-KO mice improved ventilatory function (41, 42). The systemic administration of Ad viral vectors resulted in prominent liver transduction allowing a high GAA expression in transduced hepatocytes. A single i.v. administration of Ad vector resulted in GAA production by the liver and reduced glycogen accumulation in both cardiac and skeletal muscles even in GAA-KO mice with a long-established disease (43, 44). Although a long-term persistence of Ad vectors could be achieved in the liver, an anti-hgaa humoral immune response rapidly developed, hampering maintenance of the metabolic correction (45). Using an immunodeficient GAA-KO/SCID model, a high level of hgaa secretion was obtained in the plasma for several months after Ad vector i.v. injection resulting in improved muscle strength and function, although glycogen elimination was not complete (5).

4 982 RICHARD ET AL. The availability of novel AAV serotypes has facilitated gene transfer approaches. The tropism for target tissues such as liver, striated muscle and heart is improved when vectors are pseudotyped with AAV7 (AAV2/7), AAV8 (AAV2/8), or AAV9 (AAV2/9). AAV-2/8 vectors deliver genes to the liver approximately 100-fold more efficiently than AAV2/2 vectors in adult GAA-KO mice. It is also accepted that AAV2/8 achieves high levels of liver transduction in adult mice although AAV2/1 transduces neonatal mice hepatocytes more efficiently. A single systemic delivery of AAV-1 vectors into GAA-KO neonates led to a massive transduction of heart, diaphragm, and liver that persists over 1 yr. Supraphysiological GAA expression led to a reduction of muscle glycogen and to the improvement of cardiac functions (46). The systemic injection of AAV2/8 vectors expressing hgaa into 3 month-old immunodeficient GAA-KO/SCID mice led to massive liver transduction (47, 48). The use of ubiquitous hybrid CMV enhancer and b-actin promoter to drive hgaa expression led to a marked secretion of GAA in the circulation and to a complete glycogen clearance in heart and diaphragm in males, although females had correction only in the heart (47). A similar experiment was performed using AAV2/8 and AAV2/9 vectors with muscle specific-promoters leading to a 95% and 75% glycogen reduction in heart and diaphragm, respectively (48). To increase GAA secretion efficiency, AAV2/8 vectors expressing a chimeric hgaa containing an alternative signal peptide (haat signal peptide) was constructed and injected intravenously into 3 month-old immunodeficient GAA/KO/SCID mice (49). A higher plasmatic secretion was obtained with this chimeric GAA allowing significant glycogen clearance in striated muscle, heart, diaphragm, and brain, up to 18 weeks after vector injection. Concerning the immunological consequences of the possible use of Ad and AAV vectors, it was demonstrated that liverrestricted transduction and/or expression results in an immune tolerance against rhgaa. Intravenous administration of Ad (50, 51) or AAV2/8 (52 54) vectors expressing hgaa under the control of liver-specific promoter led to a GAA expression for several months in immune competent GAA-KO mice due to evasion of immune response. Finally, even a highly persistent GAA enzymatic activity in plasma fails to achieve complete glycogen clearance and does not lead to a full reversal of pathology in skeletal muscle, especially in advanced Pompe disease (55). Because severely affected patients accumulate higher levels of glycogen than GAA-KO mice, it is unlikely that a small increase of hgaa may be sufficient to reverse the pathology. Additional therapeutic strategies should therefore be evaluated to improve GAA expression. Hematopoietic Stem Cell Gene Therapy Hematopoietic stem cell (HSC) transplantation is a potential option for lysosomal storage diseases, due to the fact that HSC are able to distribute the defective enzyme in the whole body after secretion-reuptake. HSC transplantation was tested in Pompe disease (1), but it did not improve the clinical course, probably because of a low donor hematopoietic chimerism and an inefficient GAA secretion by hematopoietic cells. However, despite significant progress in HSC transplantation, this procedure is strictly dependent of HLA-matched donor availability and is still associated with risks of graft failure or graft versus host disease (GVHD). Autologous HSC gene therapy with integrative viral vectors would improve the strategy by alleviating the GVHD and allowing high-level GAA expression/secretion from strong promoters. The efficiency of HSC-gene therapy was demonstrated in GAA-KO mice after transduction of HSC with lentiviral vectors (56, 57). The overexpression of hgaa in hematopoietic cells resulted in a major clearance of glycogen in heart, diaphragm, and liver leading to cardiac remodeling, restoration of respiratory function, as well as muscular strength improvement. Moreover, these studies demonstrated that GAA overexpression was not detrimental to hematopoietic cells and led to immune tolerance to ERT with rhgaa protein. INDUCTION OF IMMUNE TOLERANCE Patients who are unable to produce native enzyme due to deleterious mutations are named CRIM-negative patients. They are prone to develop a sustained immune response to recombinant enzyme, with a particularly high titer of anti-gaa antibodies. In GAA-KO mice, a similar immune reaction was observed after a few rhgaa injections, impairing long-term ERT studies (16). Although the role of this antibody response on ERT efficiency is unclear, it is speculated that CRIM status influences ERT outcome in infants with Pompe disease (31). Furthermore, high-dose hgaa therapy precipitated nephritic syndrome in one CRIM-negative patient, possibly due to effects of antibody complexes upon glomerular basement membrane. The induction of immune tolerance to the therapeutic enzyme would greatly enhance the benefit of ERT in these patients (58), but no immune modulation or tolerization protocol has permitted to maintain the efficacy of ERT after the formation of anti-gaa antibodies. Whereas ubiquitous expression of therapeutic proteins provoked antibody formation and cytotoxic T lymphocyte (CTL)-mediated response, liver-specific promoters induced immune tolerance to therapeutic proteins in immune competent GAA-KO mice (52 54, 58). CD4 1 CD25 1 FoxP3 1 Treg cells are critical for the development of immune tolerance to hgaa in GAA-KO mice (59). Immune tolerance induction was also demonstrated in GAA-KO mice following transplantation of hematopoietic stem cells transduced with lentiviral vectors expressing hgaa (56, 57). Immunomodulatory agents already used in clinical practice such as mycophenolate (MMF), methotrexate (MTX), and cyclosporine A/azathioprine (CsA/Aza) were evaluated in GAA-KO mice (60). The injection of MMF or CsA/Aza did not interfere with the rhgaa-specific IgG response after ERT in GAA-KO mice. On the contrary, repeated injections of MTX during the first 8 weeks of a 32 weeks

5 NEW INSIGHTS INTO THERAPEUTIC OPTIONS FOR POMPE DISEASE 983 period of ERT led to significant reduction of IgG response. It is important to note that MTX administration needs to begin at the initiation of rhgaa ERT, because once the antibody response is generated, it is refractory to MTX treatment. In a patient with an infantile form of Pompe disease, the simultaneous injection of anti-cd20 monoclonal antibody, MTX, and gamma immunoglobulin has demonstrated its capacity to drastically reduce the humoral immune response consecutive to ERT (61). Finally, it can be recommended to determine by Western-blot the CRIM status of each patient before the first infusion of recombinant enzyme. This step is essential to optimize the treatment by increasing doses and/or combining ERT with induction of immune tolerance in patients CRIM-negative and/or having a poor response. ENZYME ENHANCEMENT THERAPY Pharmacological Chaperones Mutations that affect accurate folding prevent lysosomal enzymes from reaching their final destination, the lysosome. Misfolded proteins are then recognized and targeted to the proteasome to be degraded. Specific molecules called chaperones (e.g., heat-shock proteins Bip and calnexin) support the folding of the proteins into appropriate conformation. Some mutant enzymes could maintain either full or partial catalytic properties if they acquire a correct conformation and subsequently undergo further maturation. A general strategy for lysosomal storage disorder is to search for small pharmacological chaperones that are able to bind the active site of mutant enzymes and to facilitate the folding and transport process. These molecules have the ability to improve the trafficking of the mutant protein between the endoplasmic reticulum (ER) and the lysosome, resulting in phenotypic correction (Fig. 1). Various inhibitors derived from deoxynojirimycin (DNJ) have been evaluated as pharmacological chaperones, in different lysosomal storage disorders (62). 1-deoxynojirimycin (DNJ) and N-butyl-DNJ (NBJ) were investigated in fibroblasts from Pompe patients (63, 64). A significant increase of GAA activity was observed in fibroblasts from patients carrying different GAA mutants such as L552P, G549R, Y445F, and P545L mutations. Western Blot and immunofluorescence microscopy indicated that DNJ improved the protein trafficking of numerous GAA mutants and increased mature forms in lysosomes. Interestingly, most of the GAA variants that did not respond to NBJ have amino acid substitutions in critical regions at or near the active site that presumably compromise substrate binding and catalysis, without interfering with proper folding and reaching the lysosome. These observations highlighted the fact that the nature and location of amino acid substitutions play a determinant role in the response of GAA variants to pharmacological chaperones. Furthermore, a synergistic effect of N-butyl-DNJ and rhgaa was observed in Pompe disease fibroblasts, resulting in a higher enzymatic correction due to an improved enzyme maturation and delivery to the lysosome (65). These results have important clinical implications, because the association of ERT and chaperonemediated therapy may be particularly useful in patients poorly responding to therapy. Enzyme Modification and Enhanced Delivery In some Pompe patients under ERT, an efficient reversion of the hypertrophic cardiomyopathy was observed while correction of skeletal muscle was minimal. As the uptake of exogenous rhgaa is primarily mediated by the cation-independent mannose-6-phosphate receptor (CI-MPR), the large mass of the skeletal tissue combined with the low abundance of the CI- MPR may account for this discrepancy. Moreover, rhgaa prepared from CHO cells and transgenic rabbit milk harbored a relatively low level of mannose-6-phosphate residues and have less affinity for the CI-MPR. Based on these observations, efforts were made to improve the delivery of the therapeutic enzyme to affected tissues by increasing its affinity for the CI- MPR. Enzymatic adjunction of oligosaccharides to GAA using N-acetylglucosamine-1-phosphotransferase and N-acetylglucosamine-1-phosphodiester-a-N-acetylglucosaminidase was tested (66). This hypermannose-6-phosphorylated enzyme (HP-GAA) was no more effective at clearing glycogen from Pompe mice than the unmodified enzyme, due to a sequestration by mannose receptors on endothelial cells and macrophages (66). Synthetic oligosaccharides carrying mannose-6-phosphate residues were then conjugated to the rhgaa using carbonyl/hydrazide (neorhgaa) or carbonyl/oxime-mediated chemistry (oxime-neorhgaa) (66, 67). The resulting neo-rhgaa enzyme displayed near normal specific activity, increased affinity for the CI-MPR, and was internalized 20-fold more efficiently than the naïve enzyme into myoblasts in vitro. A comparable reduction of glycogen level was obtained in heart and diaphragm using a 8-fold lower dose, and in skeletal muscle using a 4-fold lower dose. The oxime-neo-rhgaa variant displayed higher stability, increased affinity for the CI-MPR, and approximately 5-fold higher efficacy at clearing glycogen with only one-fifth the dose of unmodified enzyme (67). Interestingly, the oxime-neorhgaa generated a more rapid clearance of glycogen thereby reducing or minimizing muscle damage. These chemical modifications could significantly improve the efficacy of the treatment by reducing the enzymatic doses required and by attenuating the host immune response. Another therapeutic approach based on the use of a novel fusion protein between acid alpha glucosidase and insulin-like growth factor 2 (IGF2-GAA) has recently been developed. The product, called BMN701, using a glycosylation-independent lysosomal targeting is under trial ( NCT ). The poor accessibility of the circulating enzyme to the target tissues could play a role in the low response of skeletal muscle to ERT. Hyaluronidase (hyase) that is known to increase tissue permeability was used to facilitate delivery of rhgaa to skeletal muscle in GAA-KO mice (68). Prior intraperitoneal injection of hyase before

6 984 RICHARD ET AL. ERT administration was shown to increase GAA enzymatic activity in heart, diaphragm, and quadriceps in GAA-KO mice. SUBSTRATE REDUCTION THERAPY A synergistic therapeutic effect could be obtained by combining ERT with strategies that reduce glycogen accumulation in muscles. This concept called substrate reduction therapy (SRT) aims at decreasing the amount of storage material instead of enhancing the deficient enzymatic activity (Fig. 1). In case of Pompe disease, the muscle glycogen biosynthetic pathway should be targeted while preserving the hepatic glycogen storage in order to prevent glucose homeostasis disturbance. This approach could target either the enzymes involved in glycogen synthesis or their regulation pathway. Pioneering experiments based on the use of the RNA interference technology to inhibit the muscular isoform of glycogen synthase (GYS1) showed a dramatic decrease of lysosomal glycogen storage in myogenic cell lines and primary muscular cells from GAA-KO mice (69). The injection of an AAV-1 vector expressing the shgys1 cassette in the gastrocnemius of GAA-KO mice allowed a 50% reduction of lysosomal glycogen storage. A dual GAA/GYS1- KO was generated to study the long-term effects of ubiquitous GYS1-KO in GSDII mice (70). GAA/GYS1-KO mice exhibited a profound reduction of muscular glycogen and decreased lysosomal swelling as well as autophagic buildup by comparison with GAA-KO mice. Furthermore, cardiomegaly, muscular atrophy, and exercise capacity were significantly improved in 11- month-old GAA/GYS-1 mice. These results bring the proof of concept that modulation of glycogen synthesis could offer a novel therapeutic option for Pompe disease. Glycogen synthase is negatively regulated by phosphorylation by different kinases. Thus, GYS can be partially regulated by mtor, the mammalian target of rapamycin. mtor is a serine/threonine kinase which is part of different multiprotein complexes such as mtorc1 and mtorc2. Recently, it has been found that GAA-KO mice treated with rapamycin, an inhibitor of mtorc1, exhibited a significant decrease of muscular glycogen storage due to the phosphorylation-mediated inhibition of GYS1 (71). Furthermore, it has been demonstrated that the mtorc1 pathway regulates the muscular glycogen synthesis without affecting the liver glycogen synthesis. CONCLUSION As described here, ERT is to date the only available treatment that received approval from the authorities in most countries. Whereas impressive results were obtained with ERT, especially in infantile forms of Pompe disease, some important limitations have emerged. The financial cost of this treatment remains very high despite the efforts made to scale-up the production of the recombinant enzyme. More importantly, some biologic barriers have been highlighted, such as the immune reaction and skeletal muscle resistance. In the future, understanding of the mechanistic role of autophagy in Pompe disease may reveal novel therapeutic targets. Additional investigations are needed to get significant impact in muscular or hepatic gene targeting. As mentioned, substrate reduction therapy emerged as a relevant therapeutic option for lysosomal storage disorders, including Pompe disease. The development of modulating drugs, such as muscle sirna-targeting or modulators of regulatory pathways involved in glycogen synthesis would greatly improve the benefit of ERT. 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