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Human Molecular Genetics Advance Access originally published online on September 9, 2008
Human Molecular Genetics 2008 17(24):3876-3886; doi:10.1093/hmg/ddn290
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

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

Gaelle Douillard-Guilloux1,2, Nina Raben3, Shoichi Takikita3, Lionel Batista1,2, Catherine Caillaud1,2,* and Emmanuel Richard1,2,{dagger}

1 Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France 2 Inserm, U567, Paris, France 3 Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA

* To whom correspondence should be addressed at: Institut Cochin, Département Génétique et Développement, 24 rue du Faubourg St Jacques, 75014 Paris, France. Tel: +33 144412402; Fax: +33 144412446; Email: catherine.caillaud{at}inserm.fr

Received July 21, 2008; Accepted September 8, 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Glycogen storage disease type II (GSDII) also termed Pompe disease (MIM 232300 [OMIM] ), is an autosomal recessive disorder caused by a deficiency of the lysosomal enzyme, acid maltase or {alpha}-glucosidase (GAA; EC 3.2.1.20 [EC] ). This enzyme catalyzes the complete hydrolysis of its natural substrate glycogen by cleaving both {alpha}-1,4 and {alpha}-1,6 glucosidic linkages at an acidic pH, allowing glucose to be liberated into the cytoplasm. GAA is synthesized as a 110 kDa precursor form, which is glycosylated and phosphorylated on mannose residues in the endoplasmic reticulum and Golgi apparatus, which allows the enzyme to be transported to the lysosomes by the mannose-6-phosphate receptors (MPRs) pathway, which is common to most lysosomal enzymes. This GAA precursor can also be secreted into the extracellular space where it is taken up by the ubiquitous cell surface cation-independent MPR (CI-MPR) on surrounding or distant cells. In the lysosome, the enzyme is further modified by limited proteolysis, resulting in the formation of two mature forms of 76 and 70 kDa (1). GAA deficiency results in massive glycogen accumulation in various tissues, among which skeletal and cardiac muscle are the most affected (2). Clinically, GSDII manifests as a continuum of phenotypes; the severity of the disease largely correlates with the level of residual enzyme activity. Near complete or total loss of GAA activity results in severe myopathy and cardiomyopathy in infants leading to death before 2 years of life due to cardiorespiratory failure. Partial enzyme deficiency results in late-onset forms of GSDII with slowly progressive muscle weakness without cardiac involvement and death occurring by respiratory failure. Animal models mimicking the human disease have been created by targeted disruption of the GAA gene (3,4). The models have facilitated the development of different therapeutic approaches for GSDII. Enzyme replacement therapy (ERT) has been successfully applied for a number of lysosomal storage disorders and is now available for Pompe patients. Both preclinical and clinical studies (5–9) have shown that cardiac muscle responds well to ERT. However, most of the recombinant GAA ends up in the liver, with little or no clearing of glycogen in skeletal muscle, particularly in the advanced stage of the disease (10–12). Furthermore, a selective resistance of type II muscle fibers to ERT was demonstrated in mice, although this issue is still debated in humans (13). Other therapeutic approaches such as gene therapy have been tested in the knockout mice with different vectors. Peripheral injection of adeno-associated virus (AAV) vectors with high muscle tropism, expressing human GAA, results in efficient muscle transduction associated with sustained GAA expression and glycogen clearance in GSDII mice (14–16). However, a strong and rapid humoral immune response against human acid alpha glucosidase was observed requiring the development of immunodeficient GSDII mice models to evaluate the long-term efficiency of the treatment (17). Success of both ERT and gene therapy relies on MPR-mediated delivery of enzyme to the lysosome, a pathway which is relatively inefficient in muscle due to the low abundance of these receptors in skeletal muscle (18). Based on these observations, alternative strategies independent or complementary to ERT should be warranted.

Substrate reduction therapy (SRT) is another therapeutic approach for lysosomal storage disorders, based on the inhibition of the biosynthesis of the storage material. It is already available for type 1 Gaucher disease (19,20), while clinical trials are ongoing for type 3 Gaucher disease, Niemann-Pick disease type C and GM2 gangliosidoses (21). Therefore, we explored the feasibility of an SRT approach in the GSDII model by modulating glycogen synthesis, which requires several reactions (22). The initiation step is mediated by glycogenin (GYG; EC 2.4.1.186 [EC] ) which is a self-glycosylating protein primer that initiates glycogen granule formation (23). Then, glycogen synthase (GYS; EC 2.4.1.11 [EC] ) catalyzes the addition of glucose residues to the growing glycogen molecules through the formation of {alpha}-1,4-glucosidic linkages, and finally the branching enzyme (EC 2.4.1.18 [EC] ) forms the {alpha}-1,6-glucosidic linkages (24). Mammals express two isoforms of GYS and two of GYG, encoded by genes expressed either in muscle or liver (23–25). Extensive efforts to identify the mouse and rat versions of the liver GYG have failed and it appears that this gene may be absent in non-primates.

In this paper, we have used a ribonucleic acid (RNA) interference-based strategy to reduce glycogen synthesis in myoblasts derived from GSDII mice by modulating muscle GYS and GYG expression. An efficient inhibition of GYS and GYG messenger RNA (mRNA) was obtained, resulting in a profound decrease in lysosomal glycogen accumulation and reduction in lysosomal size in GSDII primary myoblasts. Furthermore, efficient muscle transduction with an AAV-1 vector expressing EGFP and shGYS in newborn GSDII mice resulted in strong inhibition of GYS expression and decrease in cytoplasmic and vacuolar glycogen accumulation. This substrate reduction strategy could constitute a novel approach for GSDII treatment, which may complement or be used as an alternative to ERT.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Development of self-inactivated (SIN)-lentiviral vectors expressing shRNA targeted to the muscle isoforms of GYS and GYG
Short hairpin RNA (shRNA) targeted to the murine muscle GYS (named hereafter shGYS1, shGYS2 and shGYS3) and GYG (shGYG1 and shGYG2) regions were designed (Fig. 1A). They were chosen to avoid the inhibition of hepatic glycogen synthesis (these shRNAs had low sequence homology to liver isoforms). These shRNAs were inserted into the 3' LTR (long terminal repeat) of a lentiviral construct expressing the EGFP reporter gene under the control of the ubiquitous phosphoglycerate kinase (PGK) promoter (Fig. 1C). After reverse transcription and genomic integration, two copies of the shRNA cassette were present in the provirus at both 5' and 3' LTR extremities ensuring a strong shRNA expression. Vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped vectors were produced as described (26) and titers were determined by transduction of murine myogenic C2C12 cells after serial dilutions of the vector. The infectious titers ranged from 8 x 107 to 2.8 x 108 infectious units/ml.


Figure 1
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  		Figure 1. Short hairpin RNA (shRNA) design and lentiviral constructs. (A) shRNA sequences and their position from the start codon. (B) Representation of the shRNA expressed from lentiviral constructs. (C) Schematic representation of lentiviral constructs driving shRNA expression. WPRE, woodchuck hepatitis post-transcriptional regulatory element; LTR, long terminal repeat; {Delta}U3, self-inactivated (SIN) lentiviral vector obtained after partial deletion of the U3 region; shRNAs were expressed under the control of the mammalian polymerase III H1 promoter (H1p); EGFP was expressed from the phosphoglycerate kinase (PGK) housekeeping gene promoter.

 
Efficient shRNA-mediated inhibition of GYS and GYG impairs glycogen synthesis in C2C12 myogenic cells
In order to analyze the effects of GYS and GYG inhibition on glycogen synthesis, C2C12 murine myoblasts were transduced at low (low copy, LC) or high (high copy, HC) multiplicity of infection (m.o.i.) with the lentiviral vectors (m.o.i. 2 and 50 for LC and HC, respectively). EGFP-positive (EGFP+) cells were isolated by fluorescence-activated cell-sorter (FACS) analysis 7 days after transduction followed by their expansion. Efficient gene silencing was observed in HC and LC-transduced C2C12 myoblasts as shown by real-time quantitative polymerase chain reaction (PCR) analysis of GYG and GYS mRNA expression levels (Fig. 2A and B). In HC-transduced C2C12 myoblasts, reduction in GYS and GYG mRNA reaches 89 and 75% for the most effective shGYS2 and shGYG2, respectively. A vector dose-dependent effect was observed on the inhibition efficiency, correlating with the increase of integrated proviral copy number, especially for shGYS2 (1 versus 3.4 viral copy/cell for LC versus HC shGYS2, and 1.1 versus 5.1 viral copy/cell for LC versus HC shGYG2, respectively). To assess the specificity of the shRNA constructs, several controls were performed: (i) GYS and GYG mRNA were quantified in C2C12 cells transduced with lentivectors expressing EGFP without shRNA and (ii) GYS mRNA was quantified in shGYG-transduced cells and GYG mRNA was quantified in shGYS-transduced cells. No significant gene silencing was found in these controls (data not shown).


Figure 2
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  		Figure 2. Expression of glycogen synthase (GYS) and glycogenin (GYG) messenger RNA (mRNA) in shRNA(short hairpin RNA)-transduced C2C12 cells. (A and B) GYG and GYS mRNA expression level in C2C12 myoblasts. (C and D) GYG and GYS mRNA expression level in differentiated C2C12 myotubes. Results are standardized with β-actin mRNA and expressed as a ratio versus non-transduced (NT) cells. LC and HC are cells containing low or high proviral copy number, respectively. They are expressed as the mean ± SEM of three independent experiments (for each experiment, values are the average of two samples). Differences between groups were examined using the paired t-test. *P < 0.05 compared with NT cells and EGFP-transduced C2C12 cells, #P < 0.05 HC versus LC.

 
C2C12 myoblasts expressing the most efficient shGYS2 and shGYG2 were induced to differentiate into myotubes. Cell fusion and myotube differentiation were observed in both shRNA-transduced and untransduced cells (data not shown). Both untransduced and transduced C2C12 myoblasts had low levels of glycogen ranging from 0.012 ± 0.009 to 0.017 ± 0.009 mg glycogen/mg protein. During myotube differentiation, stimulation of GYS and GYG gene expression was demonstrated by RT–PCR in untransduced cells (data not shown). The silencing of GYS and GYG genes was maintained during differentiation, with a silencing of mRNA up to 98% with shGYG2 and 95% with shGYS2 (Fig. 2C and D). Glycogen content was quantified after 140 h of differentiation (Fig. 3). In untransduced cells, glycogen increased up to 26-fold in differentiated myotubes in comparison with myoblasts, reaching 0.46 mg glycogen/mg protein. Glycogen was decreased by 50% in LC shGYS2-transduced myotubes as compared with untransduced myotubes (0.2 mg glycogen/mg protein), while glycogen was almost undetectable in HC shGYS2-transduced myotubes. Surprisingly, no effect on glycogen levels was observed in shGYG2-transduced cells (0.55 mg/mg protein). These results confirmed that GYS is a limiting step during in vitro glycogen biosynthesis in murine C2C12 myogenic cells.


Figure 3
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  		Figure 3. Glycogen content in differentiated C2C12 myotubes transduced with GYS (glycogen synthase) or GYG (glycogenin)-shRNA (short hairpin RNA). Results were expressed as mean ± SEM of four independent experiments. *P < 0.05 versus non-transduced (NT) C2C12 myotubes, #P < 0.05 versus shGYG-transduced C2C12 myotubes.

 
ShRNA-mediated inhibition of GYS and GYG results in decreased lysosomal glycogen accumulation in primary myoblasts and myotubes from GSDII mice
We then investigated whether shGYS2 and shGYG2 expression could reverse the lysosomal glycogen accumulation in myoblasts from GSDII mice (Gaa–/–). Satellite cells from normal and GSDII mice were isolated as described previously (27). Myogenic cells were transduced with shGYS2 and shGYG2-lentiviral vectors at m.o.i. 10 and 100 and EGFP+ cells were isolated by FACS. GYS and GYG mRNA levels were analyzed by quantitative RT–PCR in EGFP+ Gaa–/– primary myoblasts and differentiated myotubes. As in C2C12 cells, GYS expression was stimulated during myotube differentiation of untransduced Gaa–/– cells (Supplementary Material, Fig. S8A–C). Following transduction with shGYS2 and shGYG2-expressing vectors, the level of GYS or GYG mRNA was significantly reduced in Gaa–/– primary myoblasts (Supplementary Material, Fig. S9A and B) and myotubes (98 and 99% decreased for shGYS2 HC and shGYG2 HC, respectively) (Fig. 4A and B). The degree of inhibition was also dependent on the integrated proviral copy number in primary cells (1.3 versus 6.3 viral copy/cell for LC versus HC shGYS2, and 1.3 versus 6.0 viral copy/cell for LC versus HC shGYG2, respectively). The decrease in GYS mRNA was accompanied by a reduced expression of the GYS protein (87 and 48% for shGYS2 HC and shGYS2 LC, respectively), as compared with non-transduced myotubes (Fig. 4C). In shRNA-transduced cells, the silencing efficiency was maintained during differentiation.


Figure 4
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  		Figure 4. Short hairpin RNA (shRNA)-mediated inhibition of glycogen synthase (GYS) and glycogenin (GYG) impairs glycogen storage in murine glycogen storage disease type II (GSDII) primary myotubes. (A and B) Quantitative real-time RT–PCR analysis of GYG and GYS messenger RNA (mRNA) content in shRNA-transduced myotubes. Results are mean ± SEM of three independent experiments (values are the average of two samples). Values were standardized with ß-actin mRNA and expressed as a ratio versus non-transduced (NT) GSDII myotubes. *P < 0.05 versus NT Gaa–/– myotubes. (C) Western blot analysis of GYS expression in NT and shRNA-transduced Gaa–/– myotubes. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (D) Glycogen content in NT and shRNA-transduced Gaa–/– myotubes. *P < 0.05 versus NT Gaa–/– cells, #P < 0.05 versus Gaa+/+ myotubes.

 
As lysosomal glycogen storage is a hallmark of GSDII, the effect of shGYS2 and shGYG2 expression on the intracellular glycogen level was investigated in differentiated myotubes. Gaa–/– myotubes contained a significantly higher glycogen content as compared with Gaa+/+ after a 140 h-differentiation course (1.4 mg glycogen/mg protein versus 0.7 mg glycogen/mg protein for Gaa–/– versus Gaa+/+, respectively; P < 0.05). A significant reduction in glycogen storage (below that seen in Gaa+/+) was observed in both HC and LC shGYS and shGYG-expressing cells in differentiated Gaa–/– myotubes (0.12 mg glycogen/mg protein and 0.4 mg glycogen/mg protein for HC shGYS2 and HC shGYG2, respectively) (Fig. 4D). These results suggest that the inhibition of glycogen synthesis can prevent glycogen accumulation in GAA-deficient cells.

Enlargement of lysosomes is reversed in shGYS and shGYG-expressing cells
The enlargement and rupture of the lysosomal/endosomal compartment (due to excessive glycogen storage in lysosomes) along with autophagic buildup, are thought to be the main pathogenic mechanisms responsible for muscle damage in GSDII. Therefore, the appearance of the lysosomes was evaluated in shGYS2 and shGYG2-transduced GSDII myotubes by confocal microscopy analysis after immunostaining with LAMP1 (lysosomal-associated membrane protein 1), a late endosomal/lysosomal marker (Fig. 5). The size of lysosomes in GSDII myotubes was significantly increased as compared with wild-type (Fig. 5A and G, and B and H). The mean area of the lysosomes was 0.48 µm2 ± 0.04 versus 0.21 µm2 ± 0.02 with a maximum area of 18.6 µm2 versus 3.2 µm2 for Gaa–/– versus Gaa+/+. Periodic acid Schiff (PAS) staining of GSDII myotubes demonstrated that these enlarged lysosomes are overloaded with glycogen (data not shown). Normalization of the lysosome size was observed in Gaa–/– cells expressing HC and LC shGYS2 (Fig. 5E and K, and F and L). The maximum lysosomal area was 5.9 and 4.1 in Gaa–/– myotubes expressing LC and HC shGYS2. LC shGYG2 expression was less efficient at reducing the lysosomal hypertrophy (maximum area: 11.7) (Fig. 5C and I), but HC shGYG2 was quite effective (the maximum lysosomal area was 3.5) (Fig. 5D and J).


Figure 5
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  		Figure 5. Correction of lysosomal enlargement in shRNA (short hairpin RNA)-transduced glycogen storage disease type II (GSDII) myotubes. (A–F) Representative confocal images of fixed myotubes immunostained for lysosomal-associated membrane protein 1 (LAMP1 – late endosomal/lysosomal marker): normal (B); non-transduced (NT) GSDII (A); low-copy (C) or high-copy (D) shGYG-transduced GSDII myotubes; and low-copy (E) or high-copy (F) shGYS-transduced GSDII myotubes. (G–L) The size (expressed in µm2, X-axis) and frequency (% of total, Y-axis) of lysosomes were analyzed in normal (H), NT GSDII (G), low copy (I) or high copy (J) shGYG-transduced GSDII myotubes and low copy (K) or high copy (L) shGYS-transduced GSDII myotubes using Metamorph software.

 
AAV.shGYS2 reduces muscle glycogen storage in a murine model of GSDII
shGYS2 expression was then tested in vivo and the impact of GYS inhibition on muscle glycogen storage was evaluated in GSDII mice. High muscle tropism recombinant AAV-1 vector co-expressing both EGFP and shGYS2 (AAV.shGYS2) was developed (Fig. 6A). It was directly injected into gastrocnemius muscle of newborn GSDII mice in order to evaluate the in vivo efficacy of the shRNA-mediated therapeutic approach, using non-injected contralateral limbs as Controls. Three weeks after injection, mice were sacrificed and gastrocnemius muscles were harvested for GYS mRNA and glycogen storage analysis. EGFP microscopic visualization showed that transduction efficiency was ~100%, even though expression in individual muscle fibers was heterogeneous (Fig. 6B). The quantification of GYS mRNA revealed a 50% decrease in injected muscle as compared with non-injected contralateral muscle (Fig. 6C).


Figure 6
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  		Figure 6. Adeno-associated virus (AAV2/1) vector construction and muscle transduction efficiency in glycogen storage disease type II (GSDII) mice. (A) Schematic representation of the AAV vector construct. LITR, left inverted terminal repeat; RITR, right inverted terminal repeat; shGYS2 was expressed from the human Pol III (polymerase III) promoter H1; the cytomegalovirus (CMV) early promoter drives the EGFP expression; pA, polyadenylation signal. (B) EGFP expression on a cross-section of non-injected (left panel) and shGYS2-AAV2/1 injected (right panel) GSDII gastrocnemius muscle. (C) GYS mRNA (glycogen synthase messenger RNA) expression in non-injected and shGYS2-AAV2/1 injected GSDII gastrocnemius muscle. Results are mean ± SEM of five mice. Analyses were performed 4 weeks after muscle injection.

 
In GSDII mice, muscle glycogen was increased after 2 weeks and it was easily quantified after 3 weeks (1.2 mg/mg of protein in GSDII muscle versus 0.24 mg/mg of protein in wild-type muscle). A 62% decrease in glycogen accumulation was observed 4 weeks post-injection in AAV.shGYS2 injected muscle as compared with the contralateral muscle (0.46 mg/mg of protein), indicating that GYS inhibition can prevent in vivo glycogen accumulation (Fig. 7A).


Figure 7
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  		Figure 7. Partial metabolic correction of lysosomal glycogen storage after AAV.shGYS2 vector injection in the gastrocnemius of Gaa–/– mice. (A) Glycogen content in wild-type (Gaa+/+), non-injected and AAV.shGYS2(AAV-1 vector co-expressing both EGFP and shGYS2)-injected Gaa–/– gastrocnemius. *P < 0.05 versus wild-type muscle, #P < 0.05 versus non-injected GSDII muscle. Results are mean ± SEM of four to six mice. (B) The size (expressed in µm2, X-axis) and frequency (% of total, Y-axis) of periodic acid Schiff (PAS)-positive granules were analyzed in myofibers of injected (gray) and non-injected (black) Gaa–/– muscle cross-sections using Metamorph software. Granules of <24 µm2 were not considered because a similar distribution was found between injected and non-injected Gaa–/– muscle. (C) Cross and (D) longitudinal sections of Gaa+/+, non-injected Gaa–/– and AAV.shGYS2-injected Gaa–/– gastrocnemius were analyzed for glycogen storage after PAS staining.

 
As expected, PAS staining of untreated gastrocnemius muscle from GSDII mice demonstrated massive glycogen storage (Fig. 7C and D). Consistent with the biochemical data, a significant reduction in PAS-positive material was observed on sections from the AAV.shGYS2 injected muscle. Some myofibers had nearly normal appearance (Fig. 7C). The number and size of PAS-positive vacuoles were also greatly reduced in AAV.shGYS2 injected muscle as compared with contralateral muscle (Fig. 7B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Pompe disease or glycogenosis type II is an inherited metabolic disorder characterized by a lysosomal {alpha}-glucosidase deficiency resulting in massive glycogen storage in muscle cells. We modulated the degree of muscle glycogen accumulation by reducing the expression of the major enzymes involved in its biosynthesis and evaluated the benefits of this approach on the reversal of pathology in GSDII. Initial experiments demonstrated that differentiation of C2C12 as well as normal and GSDII primary myoblasts into multinucleate myotubes resulted in a significant increase of GYS and GYG mRNA content and subsequently a stimulation of glycogen synthesis. These results suggest that glycogen synthesis could be modulated by reducing the abundance of GYS and GYG mRNA in the cell. Therefore, an RNA interference-based strategy was used to decrease the expression of these genes. We have demonstrated that lentiviral vectors expressing shRNA targeted to murine GYS and GYG mRNA sequences, result in efficient and specific gene silencing in transduced C2C12 cells as well as in GSDII primary myoblasts. Furthermore, in the diseased cells this manipulation resulted in a significant reduction in glycogen synthesis. These data confirm that GYS and GYG are key enzymes of glycogen biosynthesis and that their inhibition is associated with severe glycogen deprivation.

However, it is important to note that glycogen synthesis is affected more by the inhibition of GYS gene expression than by the inhibition of GYG, especially in C2C12 cells. This finding could be explained by the recycling of pre-existing GYG, since even a limited amount of the initiator molecule could be sufficient to start new glycogen synthesis (28,29). Mature glycogen particles in mammalian muscle have a molecular weight between 103 and 104 kDa and can be visualized by electron microscopy (29). Even a small amount of GYG protein could store a large amount of carbohydrate. Previous reports have also speculated on the presence of alternative primers for glycogen initiation (30–32). In C2C12 cells, the initiator would either be a oligosaccharide containing {alpha}-1,4 or {alpha}-1,6 glucosyl linkages or a glucosylated protein. In Gaa–/–, where lysosomal glycogen degradation is impaired, the de novo synthesis of glycogen requires new GYG proteins. In shGYG-transduced cells, the low abundance of GYG proteins due to the degradation of GYG mRNA could be a limiting step in glycogen synthesis. Two different pathways have been hypothesized for glycogen formation: in a normal state, the pre-existing GYG or proglycogen molecules (small glycogen granules with low carbohydrate content) could be recycled whereas a de novo formation of the initiator is required during stress or supplementary needs (30,31). These data and ours suggest that glycogen storage could exist even with a low GYG level and that GYS is the best candidate to modulate glycogen synthesis and the level of glycogen.

The efficiency of the shRNAs used in this study was first assessed by the reduction in the normal amount of glycogen in C2C12. More importantly, shRNAs were efficient in the reversal of the pathological accumulation of glycogen in murine GSDII myotubes. A similar effect on glycogen accumulation was shown in vivo after the injection of AAV2/1-shGYS in the gastrocnemius of Gaa–/– mice. The area of glycogen granules was reduced by shRNA injection without altering the muscle fibrils, which suggests that inhibition of GYS could reduce the pathological enlargement of the lysosome/endosome compartment. GYS and GYG are in charge of glycogen synthesis in the cytoplasm, which contains two spatially distinct pools of glycogen, i.e. cytosolic and vacuolar (lysosomal and autophagosomal). The mechanism of glycogen uptake from the cytoplasm to the lysosome is still unknown, but strong evidence suggests that a feedback mechanism links the vacuolar and cytoplasmic glycogen (33,34). Consistent with this notion, the reduction in cytoplasmic glycogen synthesis, as shown in our experiments, is followed by a decline in the lysosomal glycogen content in GSDII myoblasts.

ERT was successfully used in treating infantile forms of Pompe disease whereas a moderate effect was observed in late-onset forms. The resistance of muscle fibers to ERT is mainly due to the low density of CI-MPR in skeletal muscle compared with cardiac muscle, and to the altered trafficking of the recombinant enzyme along the endocytic pathway (10,35). Previous studies demonstrated that fast-twitch type II myofibers [gastrocnemius, tibialis anterior (TA)] are resistant to ERT as compared with slow-twitch type I (soleus) (10–12). It has been shown that GSDII myoblasts contain a subset of late endosome/lysosomes with the acidification defect; in addition, the levels of proteins (CI-MPR, clathrin, AP-2 complex, TfR) involved in receptor-mediated endocytosis and trafficking of lysosomal enzymes are reduced in muscle cells (10,35). Although a high dosage of recombinant GAA enzyme is administered to GSDII patients during ERT, only a fraction of the enzyme is targeted to muscle lysosomes; ~80% is mistargeted and ends up in the liver (10). Therapeutic doses of recombinant human acid alpha glucosidase (rhGAA) are significantly higher than those used for therapy of other lysosomal storage diseases. For example, in Gaucher disease glucocerebrosidase is administered at a dosage of 1 mg/kg while in Pompe disease rhGAA is administered at a dosage of 20 mg/kg. Furthermore, a humoral immune response was described after ERT in both Gaa–/– mice and GSDII patients, especially in CRIM negative subjects. Raben et al. (10) showed that GSDII mice treated by ERT developed signs of anaphylaxis (such as labored breathing and collapse) and none of them survived beyond the seventh injection. The induction of immune tolerance to ERT for Pompe disease is currently under intensive investigation (36,37). Strategies aimed to decrease the therapeutic dosage of the recombinant enzyme could contribute to the safety of the treatment by reducing the immune response.

Overexpression of the muscle GYS gene in GSDII mice leads to the accumulation of abnormal polysaccharides and to a severe early-onset muscle wasting disorder (38). The knockout of muscle GYS is lethal in 90% of null pups which die soon after birth due to impaired cardiac function (39). In the surviving 10% of GYS null mice, GYS and glycogen are undetectable in cardiac and skeletal muscle. It is widely accepted that muscle is an important site for glucose disposal and one might expect that, in the absence of muscle glycogen, glucose clearance would be impaired. Surprisingly, despite impaired glucose uptake into skeletal muscle, glucose disposal is either normal or improved in muscle of GYS null mice (40). GYS-deficient mice present with normal exercise capacity suggesting that at least in mice, muscle glycogen is not essential for exercise (41). However, in humans, mutations in the GYS gene resulting in decreased muscle GYS activity have been implicated in certain diabetic populations (42). Recently, a homozygous mutation was described in the muscle GYS gene in children with severe cardiomyopathy and exercise intolerance (43). Taken together, these results indicate that a complete inactivation of the GYS enzyme in muscle can lead to unexpected adverse effects. In view of a potential clinical application of GYS modulation for GSDII, one should be aware that it is important to reduce glycogen storage without inducing major disturbances in glucose or energetic metabolism.

The aim of this study was to demonstrate the feasibility of an SRT in Pompe disease using a gene transfer approach. Further development of a hydrosoluble pharmacological agent allowing partial inactivation of GYS enzyme needs to be explored. There are several advantages associated with this approach: (i) an orally available drug could be used, allowing for a non-invasive treatment of the disease, (ii) as small molecules are non-immunogenic, host immune responses will not be induced, (iii) such a molecule does not need to be taken by the CI-MPR-mediated pathway, which is impaired in GSDII. An additional therapy that does not rely on CI-MPR-mediated delivery of the recombinant GAA may decrease lysosomal glycogen load, improve the lysosomal function, and possibly even facilitate the trafficking of the therapeutic enzyme to the lysosomes. Combined therapies may be necessary for other lysosomal diseases as well. For example, a combination of ERT (imiglucerase) with SRT (miglustat) has already demonstrated its efficacy in type 3 Gaucher patients by improving the neurological symptoms (44). In the murine model of Sandhoff disease (another lysosomal disorder affecting brain), a combination of SRT with bone-marrow transplantation enhanced the survival of the animals (45). In glycogenosis type II, involvement of the central nervous system has recently been confirmed in the murine model of the disease (46). It has been well established that recombinant GAA does not cross the blood–brain barrier. However, the small molecules (utilized in SRT) do cross the blood–brain barrier and may alleviate glycogen accumulation in the central nervous system; thus a combination of these therapeutic approaches may prove extremely beneficial. In conclusion, the approach used in this paper may become a significant component of the comprehensive therapy for GSDII.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Lentiviral vector construction and production
Different lentiviral vectors co-expressing EGFP and shRNAs, respectively, under the control of the human PGK promoter and the polymerase III (Pol III) H1 promoter were constructed. Briefly, three shRNA sequences targeting the muscular GYS mRNA (shGYS) (NM 030678) and two shRNA targeting the GYG mRNA (shGYG) (NM 013755) were designed and cloned downstream the H1 promoter using Sal1-HindIII restriction enzymes (Fig. 1A). A Xho1-Nhe1 fragment containing the H1 promoter and shRNA was introduced in the 3' U3 LTR region of the SIN-U3-PGK-EGFP-WPRE (woodchuck hepatitis post-transcriptional regulatory element) vector (kindly provided by Dr P. Charneau, Institut Pasteur, Paris, France) in order to obtain a dual shRNA expression cassette from the integrated provirus. Lentiviral vectors were produced by a triple transient transfection (26). Human kidney 293T cells were plated at a density of 5 x 106 per 10 cm tissue culture plates. The following day, they were transfected with the pCMV{Delta}8.91 packaging construct (10 µl), the VSV-G pMD.G envelope plasmid (3 µg), and the specific constructs (10 µg), using the calcium phosphate precipitation method. Thirty-six hours after the transfection, viral supernatants were collected, passed through 0.22 µm filters, centrifuged at 30 000g (90 min, 4°C) and immediately cryopreserved at –80°C. Viral titers were determined by transducing C2C12 cells with serial dilutions of viral supernatants. EGFP expression was quantified 7 days after transfection by flow cytometry analysis.

Primary culture and differentiation of myoblasts
TA muscle was removed from 4–6-week-old Gaa–/– and wild-type mice. Myoblasts were isolated as described by Ohanna et al. (27). Briefly, TA was digested for 1 h with a protease from Streptomyces griseus (Sigma-Aldrich, Saint Louis, MO, USA), followed by filtration through 100 µm nylon gauze. Cells were then plated at a low density in 12-well gelatin-coated (0.02%) plates, containing Ham F12/DMEM medium (v/v) supplemented with 20% fetal calf serum, 5 mM L-glutamine, 100 IU/ml penicillin, 100 µg streptomycin (Invitrogen, Carlsbad, CA, USA) and Ultroser G 2% (Pall Corporation, East Hills, NY, USA). Five days after, wells containing myoblasts were pooled and seeded in 6-well gelatin-coated plates. Medium was changed every 2 days and cells were trypsinized and seeded every 4 days at a density of 2–5 x 104 cells/flask (75 cm2). Myotube differentiation was induced by culturing myoblasts at a density of 3 x 105 cells/well in DMEM containing 2% horse serum (differentiation medium) in plates pre-coated with Matrigel (BD Biosciences, Bedford, MA, USA). Cells were maintained in this medium for the duration of the experiment.

Lentiviral transduction of C2C12 and primary myoblasts
Cells were transduced in 6-well plates with each viral supernatant with an estimated m.o.i. of 2 and 50 (C2C12 cells) or 10 and 100 (GSDII myoblasts), in order to select cells respectively expressing HC or LC number of shRNA. EGFP-positive cells were isolated using a FACS, and cell populations expressing either high or low level of shRNA products were isolated and expanded.

The average integrated proviral copy number was determined by real-time quantitative PCR. Total deoxyribonucleic acid (DNA) was extracted using a DNA extraction kit (Qiagen, Hilden, Germany) and amplified in the WPRE region of the SIN-U3-PGK-WPRE-shRNA integrated sequence. Samples were normalized to ß-actin DNA levels. Primers and PCR conditions were as follows: for WPRE, forward 5'-GCTATGTGGATACGCTGC-3', reverse 5'-GTTGCGGCAAACACATCA-3', annealing temperature 56°C, 2 mM MgCl2, fragment size 160 bp; for ß-actin: forward: 5'-CAGGATTCCATACCTAAGAG-3', reverse 5'-CAGGATTCCATACCTAAGAG-3'; annealing temperature 56°C, 2 mM MgCl2. The vector copy number was calculated using standards containing 0.5, 1, 2, 3, 4 and 6 copies of the WPRE plasmid.

RT–PCR analysis of GYS and GYG expression
Total RNAs from EGFP-positive C2C12 cells, primary myoblasts and muscle tissues were extracted using the mRNA mini kit (Qiagen) according to standard procedures. A quantitative real-time PCR assay was developed to detect GYS and GYG mRNA levels; ß-actin was used as an endogenous Control. PCR was carried out using a LightCycler® Carousel-Based System (Roche, Mannheim, Germany) and SYBR Green PCR Master Mix Reagent (LightCycler® RNA Amplification Kit SYBR Green, Roche). Results are expressed as a ratio versus negative Control (untransduced C2C12 or Gaa–/– primary myoblasts). Primers used were as follows: for murine muscle GYS (249 bp), forward 5'-TATCGCTGGCCGCTATGAGTT-3' and reverse 5'-CACTAAAAGGGATTCATAGAG-3'; for murine GYG (204 bp): forward 5'-ACTCAGTATTCCAAATGTGTG-3' and reverse 5'-ATCAAAACTACCTTGCTCAGA-3'; for actin (210 bp), forward 5'-GGCATAGAGGTCTTTACGG-3' and reverse 5'-GTGGCATCCATGAAACTACAT-3'.

Western blot analysis
Protein lysates from cells were prepared in radioimmunoprecipitation assay buffer containing 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS (sodium dodecyl sulfate). The following antibodies were used: rabbit anti-mouse muscle GYS monoclonal antibody, dilution: 1/500 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-mouse GAPDH (glyceraldehyde 3-phosphate dehydrogenase) monoclonal antibody, dilution: 1/3000 (Abcam, Cambridge, MA, USA), and goat anti-rabbit horseradish peroxidase monoclonal antibody, dilution: 1/3000 (BioRad, Hercules, CA, USA).

Determination of glycogen concentration
Biochemical measurement of glycogen content was performed on cultured cells. Hundred microliter of acetate buffer (0.2 M, pH 4.5) was added to frozen cells on dry ice and the mix was then heated to 90°C for 15 min and homogenized. For glycogen measurement in muscle, heart or liver, tissues were removed from mice and immediately frozen. They were then homogenized at –80°C in acetate buffer (0.2 M, pH 4.5). The cellular or tissue extracts were incubated at 54°C for 1 h in the presence or absence of Aspergillus niger amylo-{alpha}-1,4-{alpha}-1,6 glucosidase (5 U/ml; Roche, Mannheim, Germany) which converts glycogen to glucose. Samples were centrifuged and glucose level was determined in the supernatant using Glucose Assay Reagent (Sigma-Aldrich) according to the manufacturer’s instructions.

Histochemical analysis of muscle sections
Glycogen storage was determined by PAS staining. Muscle tissues were frozen and 10 µm cryosections were kept at –80°C. Sections were hydrated in deionized water for 5 min followed by incubation in 0.5% periodic acid solution at room temperature for 5 min. They were then rinsed under tap water and incubated in Schiff reagent for 15 min at room temperature. They were finally dehydrated, coverslipped in a xylene-based mounting media (Eukitt; Sigma), and analyzed by light microscopy (Nikon Eclipse E 600; Nikon, Thornwood, NY, USA).

LAMP1 staining and confocal microscopy analysis
Normal and GSDII myoblasts were plated in Permanox chamber slides (33 000 cells/cm2) for 24 h and induced to differentiate into myotubes for 8 days. Myotubes were then fixed in a 2% paraformaldehyde solution for 30 min at 37°C, followed by 1 h incubation in a blocking solution. Cells were stained with primary rat anti-mouse LAMP1 antibody (dilution 1/500; BD Pharmingen, San Diego, CA, USA), followed by AlexaFluor 680-labeled anti-rat secondary antibody (dilution 1/5000; Invitrogen). Slides were mounted in Vectashield mounting medium (Vector laboratories, Burlingame, CA, USA) and analyzed by confocal microscopy (Zeiss LSM 510 META; Zeiss, Thornwood, NY, USA).

AAV construction and injection in Gaa–/– mice
Recombinant AAV2/1 vectors co-expressing EGFP and shGYS2 respectively from the PGK promoter and the human H1 promoter were produced by the Laboratoire de Thérapie Génique (Inserm U649, Nantes, France). AAV vectors were diluted in phosphate buffer saline (PBS) and injected (6.5 x 106 UI/kg) into the gastrocnemius (left leg) of 2-day-old Gaa–/– mice (3). PBS alone was injected into the contralateral gastrocnemius (right leg) to serve as Control. Mice were sacrificed 4 weeks after injection and biochemical and histological analyses were performed on injected and non-injected muscles.

Metamorph analysis
Size of lysosomes and PAS-positive areas were determined by Metamorph analysis. Representative fields were photographed with a digital camera on a Jeol 1200 confocal microscope (JEOL, Peabody, MA, USA) and with a DXM 1200 camera on a Nikon Eclipse 600 light microscope.

Statistical analysis
All the data are presented as means ± standard error of the mean (SEM). One-way ANOVA (analysis of variance) were performed on each group. P-value <0.05 was considered as statistically significant.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This work was supported by INSERM and the Association Vaincre les Maladies Lysosomales (VML). E.R. was supported by post-doctoral fellowships from VML and the Association Française contre les Myopathies (AFM). G.D. was supported by doctoral fellowship from Genzyme (France) and AFM.


   ACKNOWLEDGEMENTS
 
We thank Dr Patrice Petit for critical reading of the manuscript. We also thank the Vector Core Facility of the University Hospital of Nantes supported by the Association Française contre les Myopathies for providing the AAV vectors.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} Present address: INSERM U876, IFR 66, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux; Email: emmanuel.richard{at}u-bordeaux2.fr Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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G. Douillard-Guilloux, N. Raben, S. Takikita, A. Ferry, A. Vignaud, I. Guillet-Deniau, M. Favier, B. L. Thurberg, P. J. Roach, C. Caillaud, et al.
Restoration of muscle functionality by genetic suppression of glycogen synthesis in a murine model of Pompe disease
Hum. Mol. Genet., February 15, 2010; 19(4): 684 - 696.
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