Glucosidase Uptake in the Limb Muscles of Mice With Pompe Disease

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1 Adjunctive β2-agonist Treatment Reduces Glycogen Independently of Receptor-Mediated Acid α- Glucosidase Uptake in the Limb Muscles of Mice With Pompe Disease Benjamin Farah, BS 1, Lauran Madden, PhD 2, Songtao Li 3,Sierra Nance 3,4, Andrew Bird, BA 3, Nenad Bursac, PhD 2, Paul M Yen, MD 1, Sarah P Young, PhD 3, and Dwight D Koeberl, MD, PhD 3. 1 Cardiovascular and Metabolic Disease Program, Duke-NUS Graduate Medical School, Singapore, Singapore, ; 2 Department of Biomedical Engineering, Duke University, Durham, NC, United States, 27710; 3 Division of Medical Genetics, Department of Pediatrics, Duke University Medical Center, Durham, NC, United States, 27710; 4 current address: Winston Salem State University, Winston-Salem, North Carolina

2 Page 2 NONSTANDARD ABBREVIATIONS acid α-glucosidase (GAA) adeno-associated virus (AAV) cation-independent mannose-6-phosphate receptor (CI-MPR) knockout (KO) double knockout (DKO)

3 Page 3 Abstract Enzyme or gene replacement therapy with acid α-glucosidase (GAA) has achieved only partial efficacy in Pompe disease. We evaluated the effect of adjunctive clenbuterol treatment upon cation-independent mannose-6-phosphate receptor (CI-MPR) mediated uptake and intracellular trafficking of GAA during muscle-specific GAA expression with an adeno-associated virus (AAV) vector in GAA knockout (KO) mice. Clenbuterol, which increases expression of CI-MPR in muscle, was administered with the AAV vector. This combination therapy increased latency during Rotarod and wirehang testing at 12 weeks, in comparison with vector alone. The mean urinary glucose tetrasaccharide (Glc4), a urinary biomarker, was lower in GAA- KO mice following combination therapy, compared with vector alone. Similarly, glycogen content was lower in cardiac and skeletal muscle following 12 weeks of combination therapy in heart, quadriceps, diaphragm, and soleus, compared with vector alone. These data suggested that clenbuterol treatment enhanced trafficking of GAA to lysosomes, given that GAA was expressed within myofibers. The integral role of CI- MPR was demonstrated by the lack of effectiveness from clenbuterol in GAA-KO mice that lacked CI-MPR in muscle, where it failed to reverse the high glycogen content of the heart and diaphragm or impaired wirehang performance, However, the glycogen content of skeletal muscle was reduced by the addition of clenbuterol in absence of CI-MPR, as was lysosomal vacuolation, which correlated with increased AKT signaling. In summary, β2-agonist treatment enhanced CI-MPR mediated uptake and trafficking of GAA in mice with Pompe disease, and a similarly enhanced benefit might be expected in other lysosomal storage disorders. Keywords: Mannose-6-phosphate receptor, gene therapy, adeno-associated virus, acid alpha-glucosidase, acid maltase, glycogen storage disease type II

4 Page 4 Introduction Enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (GAA) has prolonged ventilator-free survival and muscle strength in patients with Pompe disease; however, the limitations of ERT have become increasingly evident and many patients eventually become ventilator-dependent. The enzyme dosages required for ERT in Pompe disease range up to 100-fold greater than those for other lysosomal disorders, which can be attributed at least in part to the large muscle mass and to the formation of anti-gaa antibodies. ERT depends upon the uptake of recombinant lysosomal enzymes by the cation-independent mannose-6-phosphate receptor (CI-MPR) at the plasma membrane and trafficking to the lysosomes, and CI-MPR is present at low levels in the skeletal muscle postnatally (1). Clenbuterol treatment has been previously shown to enhance the biochemical correction of muscle from ERT in GAA (KO) mice with Pompe disease (2); furthermore, clenbuterol at a lower dose and a second β2- agonist (albuterol) increased Rotarod latency and biochemical correction following β 2 -agonist treatment, in comparison with ERT alone (3). Glycogen content was reduced significantly in all striated muscles evaluated with the exception of tibialis anterior by clenbuterol treatment, in comparison with ERT alone (3). Both of the β 2 -agonists evaluated increased uptake of rhgaa in the cerebral and cerebellar hemispheres of the brain during ERT, and glycogen storage was reduced accordingly in the brain (3). Furthermore, Western blot analysis revealed that clenbuterol treatment increased the signal for CI-MPR in the extensor digitalis longus (EDL) muscle, cerebellum, and cerebrum (3). The effect of CI-MPR upon gene therapy has been evaluated by administering an adeno-associated virus (AAV) vector encoding human GAA driven by a liver-specific regulatory cassette (AAV-LSPhGAA) to establish a liver depot for GAA secretion. A low dose of AAV-LSPhGAA, 2x10 10 vector particles (vp), was injected intravenously to GAA-KO mice +/- clenbuterol treatment (4). The effect of clenbuterol was evident,

5 Page 5 when Rotarod testing increased by 75% following vector administration and clenbuterol treatment, in comparison with vector administration alone. The effect of clenbuterol was further demonstrated by increased biochemical correction in striated muscles and the brain from this combination therapy, in comparison with the vector alone (4). These data demonstrated a synergistic effect upon efficacy from AAV vector administration and clenbuterol treatment in mice with Pompe disease. The direct transduction of non-dividing myofibers has a distinct advantage for the treatment of a myopathy such as Pompe disease, especially for AAV vectors that remain almost exclusively episomal and are lost from dividing hepatocytes in the liver (5, 6). Muscle-restricted transgene expression with an AAV vector containing a muscle-specific regulatory cassette significantly reduced glycogen accumulations in the heart and skeletal muscle, while evading the transgene-directed T cell responses otherwise directed against GAA expressed with a constitutive regulatory cassette in adult GAA-KO mice (7, 8). However, the dosage requirements to achieve biochemical correction of skeletal muscle were approximately 10-fold higher with the muscle-expressing vector, in comparison with the liver-expressing vector (8, 9). Therefore, we have evaluated the effect of adjunctive β 2 -agonist therapy in combination with the muscle-expressing vector, thereby potentially reducing vector dosage requirements for the AAV vector by augmenting the trafficking of GAA to lysosomes. Materials and Methods Preparation of AAV vectors The vector, AAV-MHCK7hGAApA contains the MHCK7 regulatory cassette, the human GAA cdna, and a human growth hormone polyadenylation sequence flanked by the AAV2 terminal repeats (8). Briefly, 293 cells were transfected with an AAV vector plasmid, the AAV packaging plasmid (10) (courtesy of Dr. James M. Wilson, University of Pennsylvania, Philadelphia, PA), and padhelper (Stratagene, La Jolla, CA). Cell

6 Page 6 lysate was harvested 48 hours following infection and freeze-thawed 3 times, and isolated by sucrose cushion pelleting followed by two cesium chloride gradient centrifugation steps. AAV stocks were dialyzed against 3 changes of Hanks buffer, and aliquots were stored at -80 o C. The number of vector DNA containing-particles was determined by DNase I digestion, DNA extraction, and Southern blot analysis. All viral vector stocks were handled according to Biohazard Safety Level 2 guidelines published by the NIH. Generation of muscle-specific CI-MPR-KO and DKO mouse models: CI-MPR-KO mice were generated using a muscle-specific promoter (muscle creatine kinase; CK) and the cre/loxp conditional knock out system as described previously (11). The muscle-specific CI-MPR-KO mice were crossed with GAA-KO mice to generate muscle-specific DKO mice. This mouse colony was subsequently screened to be GAA -/-, M6PR flox/flox and MCK-Cre positive. DKO mice were genotyped and bred as described (2). At the indicated time points post-injection, tissue samples were obtained and processed as described below. All animal procedures were done in accordance with Duke University Institutional Animal Care and Use Committee-approved guidelines. In vivo evaluation of AAV vector-mediated efficacy Clenbuterol (30 mg/l) was provided continuously from day 1 in the drinking water, and vector as administered on day 1 by intravenous injection. Rotarod testing was performed as described (12). Wirehang testing was performed with a 0.5 cm mesh hardware cloth fixed to an 8 by 10 inch frame. Mice were placed on the wire mesh, which was slowly inverted 6 inches over a cage containing paper bedding. The latency, or time until the mouse fell off of the wire mesh, was recorded. GAA activity and glycogen content were analyzed as described (12). Histologic processing and staining of brain was performed using a modified paraffin processing and staining protocol as previously described (13). Quantification of vector DNA was performed as follows using primers for human GAA and mouse β-actin (14). Plasmid DNA corresponding to 0.01 copy to 10 copies of human GAA gene (in 500 ng genomic DNA) was used in a standard curve.

7 Page 7 LAMP2 detection by immunohistochemistry Paraffin embedded sections were de-paraffinized, rehydrated, then blocked in PBS containing 5% chick serum and 0.1% Triton-X 100. Primary antibody for LAMP-2 (H4B4, Santa Cruz, 1:50) was applied overnight at 4 C. Chicken anti-mouse conjugated 488 (Invitrogen, 1:200) was used for detection and DAPI for counterstaining of nuclei. Five 40x images were acquired per slide for blinded counting of LAMP-2 positive muscle fibers. Western Blotting Protein was prepared by homogenization and sonication, as described for quantification of GAA and glycogen. From the protein samples, an aliquot was taken and protein concentration determined by BCA Kit (Bio-Rad). To the remainder of the sample was added Laemmli sample buffer (250 mmol/l Tris, ph 7.4, 2% w/v sodium dodecyl sulfate, 25% v/v glycerol, 10% v/v 2-mercaptoethanol, and 0.01% w/v bromophenol blue), followed by heating to 105 C for 5 minutes, and storage at -80 C. Equal amounts of each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immediately transferred to a polyvinylidene difluoride membrane (Bio-Rad) using Towbin transfer buffer (25 mmol/l Tris, ph 8.8, 192 mmol/l glycine, 15% v/v methanol). Following transfer, membranes were blocked in 5% milk in PBST. Primary antibodies (AKT #2938, LC3B #2775, SQSTM1/p62 #5114, GAPDH #D16H11 from Cell Signalling Technologies) were added to the membranes in 1% w/v bovine serum albumin in PBST and incubated overnight at 4 C. Membranes were washed 3 times in PBST, followed by incubation in anti-rabbit HRP conjugated secondary antibody (Sant Cruz Biotechnology Inc.) in 1% w/v bovine serum albumin in PBST for 1 hr at room temperature. Blots were again washed 3 times in PBST, and then once in PBS. Blots were developed with enhanced chemiluminescence system (GE Healthcare), and images acquired on a GelDoc XR+ imager (Bio-Rad).

8 Page 8 Densitometry Western blot bands were quantified using ImageJ software (NIH). LC3 turnover was analyzed by comparing the intensity of the LC3-II band to the internal control GAPDH, as is currently recommended practice.(1) Results were shown as mean ± SEM. Significance between conditions (p<0.05) was ascertained by twotailed t-test. Results Treatment with clenbuterol has increased CI-MPR expression in muscle accompanied by increased CI-MPR mediated uptake of GAA and trafficking to the lysosome, where GAA was activated to reduce lysosomal glycogen (Figure 1A). In order to study intracellular trafficking of GAA, a muscle-expressing AAV vector as pseudotyped as AAV8 (AAV2/8-MHCK7hGAApA) in a 5-fold lower quantity than was previously required to clear glycogen storage from the heart (8) was administered intravenously to adult GAA-KO mice (2x10 11 vp). Biochemical efficacy was demonstrated by increased GAA activity in skeletal muscle at 12 weeks following vector administration, in comparison with untreated GAA-KO mice (Figure 1B). Clenbuterol further elevated GAA activity in the heart and diaphragm, in comparison with the vector alone. The combination therapy increased GAA activity to greater than the level observed in wildtype mice; however, GAA activity remained lower than wildtype levels in skeletal muscle (Figure 1B). Vector administration reduced the glycogen content of the diaphragm, soleus, and EDL (Figure 1C). The addition of clenbuterol significantly reduced glycogen content in the heart and all skeletal muscles evaluated, in comparison with the vector alone. However, all groups of GAA-KO mice had higher glycogen content than wildtype mice (<0.1 μm glucose/mg protein). The mean urinary glucose tetrasaccharide (Glc 4 ) biomarker of glycogen storage and turnover was significantly lower in GAA-KO mice following combination treatment compared with vector-only treated mice (Figure 1D). Urinary Glc 4 correlated with heart glycogen content (R 2 = 0.72) and not with muscle glycogen

9 Page 9 (Figure 1E), indicating a major effect upon the heart. These data suggested that the β2 agonist treatment enhanced trafficking of GAA to lysosomes and activation within striated muscle, without increasing receptormediated uptake per se, in the context of muscle-specific GAA expression. The dependence of efficacy from clenbuterol upon CI-MPR expression was evaluated in CI-MPR-KO/GAA- KO mice (double-ko/dko mice) that lacked CI-MPR expression in muscle. GAA activity was only slightly increased by the addition of clenbuterol in diaphragm, while GAA activity remained unchanged in the heart and limb muscles (Figure 2A). Glycogen content was only slight reduced in the quadriceps by the addition of clenbuterol, and unchanged in the heart and diaphragm (Figure 2B). Thus, the additional biochemical efficacy from clenbuterol administration seemed to depend upon CI-MPR expression, because GAA activity remained the same or increased only slightly following clenbuterol administration. The addition of clenbuterol had no effect upon glycogen storage in the heart and diaphragm of DKO mice; however, glycogen content was slightly reduced in all of the limb muscles by clenbuterol, which raised the possibility of an additional mechanism for its activity (Figure 2B). Vector DNA quantification was performed in heart to determine whether variability in transduction might underlie the differences between groups of mice; however, no significant differences were observed between any of the treatment groups, indicating equivalent transgene delivery in all groups (Figure 2C). The effect of adjunctive clenbuterol upon muscle function was investigated by performing Rotarod and wirehang testing 12 weeks following vector administration in GAA-KO and DKO mice (Figure 3). Improved function on the wirehang test in GAA-KO mice only, not DKO mice, indicated dependence upon CI-MPR for an increase in muscle strength. However, the Rotarod latency increased for both GAA-KO and DKO mice following clenbuterol treatment, revealing beneficial roles of clenbuterol on this type of neuromuscular function even in absence of CI-MPR expression. Previously clenbuterol increased Rotarod performance in absence of ERT in GAA-KO, consistent with an effect of muscle hypertrophy 2). The wirehang latency of

10 Page 10 vector-injected DKO mice was markedly lower, in comparison with vector-injected GAA-KO mice (Figure 3), consistent with the demonstrated lack of efficacy from AAV vector administration in DKO mice (4). Given the partial biochemical response of the quadriceps and other limb muscles in absence of CI-MPR expression, LAMP2 vacuolization was analyzed as a marker for glycogen accumulation (Figure 4A). As expected, greater LAMP-2 expression was observed in GAA-KO mice compared to wild-type mice (15). Although vector treatment alone did not significantly decrease LAMP-2 expression, the addition of clenbuterol significantly reduced LAMP-2 expression (Figure 4B), which resembled that observed in wildtype animals (Figure 4A). A dysregulation of macroautophagy, a process by which cytosolic components are sequestered and delivered to lysosomes, has long been implicated in the pathology of Pompe's disease (16). Pompe disease is associated with an increase in the number of autophagosomes and a decrease in autophagic flux, as the lysosomal degradation of autophagosomal targets is impaired (17). We thus investigated whether vector administration affected this abnormal autophagy [Figure 5]. Treatment with the AAV vector significantly decreased the protein levels of LC3-II, a marker for autophagosomal formation (Figure 5A and 5C) (18). Moreover, this decrease was associated with an increase in autophagic flux, as the treatment also significantly decreased the abnormal accumulation of SQSTM1/p62 (Figure 5A and 5B), implying an increase in autophagic flux (19). Adjunctive treatment with clenbuterol caused a non-statistically significant increase in the levels of both LC3-II and SQSTM1/p62 compared with vector alone; however, when compared to the dramatic improvement seen in the clenbuterol-treated mice, it likely was not biologically significant. Furthermore, we investigated the AKT/mTOR pathway, which is known to inhibit autophagy (20). GAA-KO mice showed increased phosphorylation of AKT (Figure 6A-C), and the mtor substrate p70s6k (Figure 6D-F), compared to the wild type mice. Vector administration significantly reduced both of these phosphorylations, which were further reduced with adjunctive clenbuterol treatment (Table). Of note, the

11 Page 11 reduction of AKT/mTOR signaling by clenbuterol did not seem to correlate with decreased p62 in the clenbuterol-treated mice (Table), implying that any clenbuterol effect was mediated by a different pathway. Discussion The current study was designed to address the question of whether the increased CI-MPR expression associated with clenbuterol administration might improve the trafficking of GAA to lysosomes in mice with Pompe disease, given the low expression of CI-MPR in striated muscle postnatally (1). The addition of clenbuterol following AAV vector-mediated GAA expression in muscle also reduced glycogen content in the heart and skeletal muscle. Muscle strength also was enhanced by adjunctive therapy with clenbuterol, similar to the biochemical correction that occurred in muscle. The effect of clenbuterol was mostly dependent upon CI-MPR expression, consistent with the role of increased CI-MPR in mediating efficacy during GAA replacement in Pompe disease (2). Unexpectedly, glycogen content in skeletal muscle was slightly reduced in concert with clenbuterol-increased Rotarod performance in DKO mice that lacked CI- MPR in muscle. The reduction in LAMP-2, observed in both the GAA-KO and DKO mouse strains, correlated with the reduction in glycogen content similar to that observed with ERT treatment (1). Previously, clenbuterol slightly reduced the glycogen content of skeletal muscle by itself (3), which presumably did not depend upon CI-MPR expression in absence of GAA replacement. Hence, clenbuterol proved to have both CI-MPR dependent and independent benefits during muscle-targeted gene therapy in mice with Pompe disease. One of the hallmarks of Pompe disease is an abnormal increase in autophagosomes. It was originally suspected that an increase in autophagy was responsible for the pathology of Pompe disease, but further research showed that there was actually a functional deficit in autophagy, despite the increased number of autophagosomes (17). Although the exact link between the block in autophagic flux and the pathology of Pompe's disease remains unclear, recent work has shown that restoration of autophagic flux following ERT correlated with improved muscle function in patients with Pompe disease (21). The improvement in function

12 Page 12 and autophagic flux following AAV vector administration agreed with this earlier finding. Clenbuterol, although further improving function, does not seem to further improve the autophagic flux, implying that its action is through a separate pathway. The increase in AKT and p70s6k in the GAA-KO mice that occurred after recovery from AAV treatment (Figure 6) was an unexpected finding. Although we initially predicted that AKT levels should be higher in the treated mice, secondary to increased muscle mass and IGF-1 signaling (22), the opposite trend was observed. Only one previous study has investigated the role of AKT/mTOR in Pompe's disease, when Nishiyama and colleagues found AKT signaling to be reduced in the fibroblasts of affected patients, with improvement in lysosomal delivery of GAA following activation of this pathway by insulin (23). Although the results from our study seem to contradict these previous findings, it is important to note that fibroblasts and skeletal muscle may respond differently to the loss of GAA, and AKT may play different roles in different tissues. Another interesting finding is that the LC3-II levels were highly increased (Figure 5C) in the GAA- KO mice, which also showed increased mtor signaling (Figure 6). While mtor has classically been known to inhibit the first step of autophagosomal formation, leading to a decrease in LC3-II protein levels (20), a recent study has shown that it may also inhibit lysosomal activity as well (24), and that effect may be predominating in this system. In our study, AAV vector transduction reduced the increased AKT signaling in GAA-KO mice, and the addition of clenbuterol further reduced pakt (Table). Thus, the effect of adding clenbuterol was to reduce the abnormalities of autophagy further, in comparison with partial correction from vector administration alone. Moreover, clenbuterol and AAV transduction further enhanced clearance of glycogen in limb muscles above the amount observed from AAV transduction in both GAA-KO and DKO mice. The observation that clenbuterol treatment reduced glycogen storage in DKO mice confirms that clenbuterol works independently of GAA to significantly reduce glycogen content in the skeletal muscles of mice with Pompe disease (3), because AAV transduction with GAA was ineffective in DKO mice (Figure 2) as described (4). Furthermore, clenbuterol s observed effects on reducing mtor signaling and glycogen

13 Page 13 content were similar to the reported effect of rapamycin, which inhibited mtor and reduced glycogen storage in the skeletal muscle of GAA-KO mice (25). Adjunctive therapy with clenbuterol was beneficial in combination with ERT or gene therapy (2-4); however, clinical translation of this strategy will depend upon the response of humans to clenbuterol. One critical factor will be the effective serum concentration of β2-agonists. Limited data are available, but the effective concentration (EC50) for clenbuterol was lower for humans and other higher mammals than for rodents. For example, the EC50 for clenbuterol for the relaxation of rat smooth muscle was approximately 10-fold higher than the EC50 for equine or human smooth muscle (26-28). Moreover, the standard dosages of clenbuterol used clinically have increased muscle strength or increased muscle mass in several studies. Patients with chronic heart failure developed increased lean muscle mass after taking clenbuterol at 80 μg/day for 12 weeks (29). Patients with amyotrophic lateral sclerosis (ALS) were stronger as determined by the myometer score for upper and lower extremities after taking clenbuterol, 60 μg /day, for 3 months (30). A small group of patients with Duchenne muscular dystrophy developed increased power and volume of well-preserved muscle following 18 months of taking clenbuterol, μg/day (31). Therefore, standard dosages of clenbuterol might be beneficial in Pompe disease by increasing muscle strength, possibly through biochemical correction and improvement in autophagy (Table). Our data also raise the intriguing possibility of further improving the treatment by using an mtor inhibtor such as rapamycin in conjunction with clenbuterol as well as gene therapy. Previous work has shown a beneficial effect by gene therapy in combination with rapamycin (25), as mtorc1 inhibition reduces glycogen synthesis. Additionally, mtorc1 inhibition increases the activity of TFEB (32), a transcription factor known as the master regulator of autophagosomal and lysosomal biosynthesis (33). More intriguingly, Spampanato et. al. have recently shown that TFEB overexpression leads to direct benefit in Pompe's

14 Page 14 disease models, not only by improving lysosomal function, but also through a new form of exocytosis in which autolysosomal glycogen is excreted from the cell (34). Although long-term rapamycin therapy can cause immunosupression (35) and insulin resistance (36), these potential side effects, may be acceptable given the severe phenotype of Pompe's disease. The development of new mtor inhibitors also may reduce some of these adverse effects. Overall, our results suggest that the therapeutic role of clenbuterol can be expanded from the concept of enhanced receptor-mediated uptake of GAA to include enhanced trafficking of muscle-expressed GAA during gene therapy. Furthermore, its effects on autophagy, possibly through AKT signaling indicate the potential benefit from a second mechanism independent of CI-MPR in limb muscle. Ultimately, clenbuterol could be beneficial in patients with late-onset Pompe disease for whom ERT is not an option, because it could reduce glycogen storage in limb muscles and result in positive effects upon ambulation. ACKNOWLEDGEMENTS This work was supported by NIH Grant R01 HL from the National Heart, Lung, and Blood Institute, and by NIH Grant R01AR from the National Institute of Arthritis and Musculoskeletal and Skin Disorders. Muscle specific CI-MPR-KO mice were provided courtesy of Dr. Randy Jirtle, Duke University (Durham, NC). RhGAA was provided under agreement with Genzyme Corporation. The AAV8 packaging plasmid was provided courtesy of Dr. James M. Wilson at the University of Pennsylvania (Philadelphia, PA). Conflict of interest: DB, DDK, and SPY have received research/grant support from Genzyme Corporation in the past.

15 Page 15 REFERENCES 1. Raben, N., Danon, M., Gilbert, A. L., Dwivedi, S., Collins, B., Thurberg, B. L., Mattaliano, R. J., Nagaraju, K., and Plotz, P. H. (2003) Enzyme replacement therapy in the mouse model of Pompe disease. Mol Genet Metab 80, Koeberl, D. D., Luo, X., Sun, B., McVie-Wylie, A., Dai, J., Li, S., Banugaria, S. G., Chen, Y. T., and Bali, D. S. (2011) Enhanced efficacy of enzyme replacement therapy in Pompe disease through mannose-6-phosphate receptor expression in skeletal muscle. Mol Genet Metab 103, Koeberl, D. D., Li, S., Dai, J., Thurberg, B. L., Bali, D., and Kishnani, P. S. (2012) beta2 Agonists enhance the efficacy of simultaneous enzyme replacement therapy in murine Pompe disease. Mol Genet Metab 105, Li, S., Sun, B., Nilsson, M. I., Bird, A., Tarnopolsky, M. A., Thurberg, B. L., Bali, D., and Koeberl, D. D. (2013) Adjunctive beta2-agonists reverse neuromuscular involvement in murine Pompe disease. FASEB J 27, Schnepp, B. C., Clark, K. R., Klemanski, D. L., Pacak, C. A., and Johnson, P. R. (2003) Genetic fate of recombinant adeno-associated virus vector genomes in muscle. J Virol 77, McCarty, D. M., Young, S. M., Jr., and Samulski, R. J. (2004) Integration of adeno-associated virus (AAV) and recombinant AAV vectors. Annu Rev Genet 38, Sun, B., Zhang, H., Franco, L. M., Brown, T., Bird, A., Schneider, A., and Koeberl, D. D. (2005) Correction of glycogen storage disease type II by an adeno-associated virus vector containing a muscle-specific promoter. Mol Ther 11, Sun, B., Young, S. P., Li, P., Di, C., Brown, T., Salva, M. Z., Li, S., Bird, A., Yan, Z., Auten, R., Hauschka, S. D., and Koeberl, D. D. (2008) Correction of multiple striated muscles in murine Pompe disease through adeno-associated virus-mediated gene therapy. Mol Ther 16, Franco, L. M., Sun, B., Yang, X., Bird, A., Zhang, H., Schneider, A., Brown, T., Young, S. P., Clay, T. M., Amalfitano, A., Chen, Y. T., and Koeberl, D. D. (2005) Evasion of immune responses to

16 Page 16 introduced human acid alpha-glucosidase by liver-restricted expression in glycogen storage disease type II. Mol Ther 12, Gao, G. P., Alvira, M. R., Wang, L., Calcedo, R., Johnston, J., and Wilson, J. M. (2002) Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Nat Acad Sci USA 99, Wylie, A. A., Pulford, D. J., McVie-Wylie, A. J., Waterland, R. A., Evans, H. K., Chen, Y. T., Nolan, C. M., Orton, T. C., and Jirtle, R. L. (2003) Tissue-specific inactivation of murine M6P/IGF2R. Am J Pathol 162, Sun, B., Zhang, H., Franco, L. M., Young, S. P., Schneider, A., Bird, A., Amalfitano, A., Chen, Y. T., and Koeberl, D. D. (2005) Efficacy of an adeno-associated virus 8-pseudotyped vector in glycogen storage disease type II. Mol Ther 11, Taksir, T. V., Griffiths, D., Johnson, J., Ryan, S., Shihabuddin, L. S., and Thurberg, B. L. (2007) Optimized preservation of CNS morphology for the identification of glycogen in the Pompe mouse model. J Histochem Cytochem 55, Zhang, P., Sun, B., Osada, T., Rodriguiz, R., Yang, X. Y., Luo, X., Kemper, A. R., Clay, T. M., and Koeberl, D. D. (2012) Immunodominant liver-specific expression suppresses transgene-directed immune responses in murine pompe disease. Hum Gene Ther 23, Raben, N., Fukuda, T., Gilbert, A. L., de Jong, D., Thurberg, B. L., Mattaliano, R. J., Meikle, P., Hopwood, J. J., Nagashima, K., Nagaraju, K., and Plotz, P. H. (2005) Replacing acid alphaglucosidase in Pompe disease: Recombinant and transgenic enzymes are equipotent, but neither completely clears glycogen from type II muscle fibers. Mol Ther 11, Engel, A. G. (1970) Acid maltase deficiency in adults: studies in four cases of a syndrome which may mimic muscular dystrophy or other myopathies. Brain 93, Raben, N., Hill, V., Shea, L., Takikita, S., Baum, R., Mizushima, N., Ralston, E., and Plotz, P. (2008) Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease. Hum Mol Genet 17,

17 Page Klionsky, D. J., Abdalla, F. C., Abeliovich, H., Abraham, R. T., Acevedo-Arozena, A., Adeli, K., Agholme, L., et al. (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, Masiero, E., Agatea, L., Mammucari, C., Blaauw, B., Loro, E., Komatsu, M., Metzger, D., Reggiani, C., Schiaffino, S., and Sandri, M. (2009) Autophagy is required to maintain muscle mass. Cell Metab 10, Alers, S., Loffler, A. S., Wesselborg, S., and Stork, B. (2012) Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol Cell Biol 32, Nascimbeni, A. C., Fanin, M., Masiero, E., Angelini, C., and Sandri, M. (2012) Impaired autophagy contributes to muscle atrophy in glycogen storage disease type II patients. Autophagy 8, Bonaldo, P., and Sandri, M. (2013) Cellular and molecular mechanisms of muscle atrophy. Dis Model Mech 6, Nishiyama, Y., Shimada, Y., Yokoi, T., Kobayashi, H., Higuchi, T., Eto, Y., Ida, H., and Ohashi, T. (2012) Akt inactivation induces endoplasmic reticulum stress-independent autophagy in fibroblasts from patients with Pompe disease. Mol Genet Metab 107, Zhou, J., Tan, S. H., Nicolas, V., Bauvy, C., Yang, N. D., Zhang, J., Xue, Y., Codogno, P., and Shen, H. M. (2013) Activation of lysosomal function in the course of autophagy via mtorc1 suppression and autophagosome-lysosome fusion. Cell Res 23, Ashe, K. M., Taylor, K. M., Chu, Q., Meyers, E., Ellis, A., Jingozyan, V., Klinger, K., Finn, P. F., Cooper, C. G., Chuang, W. L., Marshall, J., McPherson, J. M., Mattaliano, R. J., Cheng, S. H., Scheule, R. K., and Moreland, R. J. (2010) Inhibition of glycogen biosynthesis via mtorc1 suppression as an adjunct therapy for Pompe disease. Mol Genet Metab 100, Iizuka, H., Osaka, Y., Kondo, S., and Morita, T. (1998) Effect of an atypical adrenergic beta3- agonist, GS-332: sodium (2R)-[3-[3-[2-(3-chlorophenyl)-2-hydroxyethylamino]cyclohexyl]phenoxy] acetate, on urinary bladder function in rats. J Smooth Muscle Res 34,

18 Page Torneke, K., Ingvast Larsson, C., and Appelgren, L. E. (1998) A comparison between clenbuterol, salbutamol and terbutaline in relation to receptor binding and in vitro relaxation of equine tracheal muscle. J Vet Pharmacol Ther 21, Nials, A. T., Coleman, R. A., Johnson, M., Magnussen, H., Rabe, K. F., and Vardey, C. J. (1993) Effects of beta-adrenoceptor agonists in human bronchial smooth muscle. Br J Pharmacol 110, Kamalakkannan, G., Petrilli, C. M., George, I., LaManca, J., McLaughlin, B. T., Shane, E., Mancini, D. M., and Maybaum, S. (2008) Clenbuterol increases lean muscle mass but not endurance in patients with chronic heart failure. J Heart Lung Transplant 27, Soraru, G., Pegoraro, E., Spinella, P., Turra, S., D'Ascenzo, C., Baggio, L., Mantovan, M. C., Vergani, L., and Angelini, C. (2006) A pilot trial with clenbuterol in amyotrophic lateral sclerosis. Amyotroph Lateral Scler 7, Oya, Y., Morita, H., Ogawa, M., Nonaka, I., Tsujino, S., and Kawai, M. (2001) [Adult form of acid maltase deficiency presenting with pattern of muscle weakness resembling facioscapulohumeral dystrophy]. Rinsho Shinkeigaku 41, Laplante, M., and Sabatini, D. M. (2013) Regulation of mtorc1 and its impact on gene expression at a glance. J Cell Sci 126, Sardiello, M., Palmieri, M., di Ronza, A., Medina, D. L., Valenza, M., Gennarino, V. A., Di Malta, C., Donaudy, F., Embrione, V., Polishchuk, R. S., Banfi, S., Parenti, G., Cattaneo, E., and Ballabio, A. (2009) A gene network regulating lysosomal biogenesis and function. Science 325, Spampanato, C., Feeney, E., Li, L., Cardone, M., Lim, J. A., Annunziata, F., Zare, H., Polishchuk, R., Puertollano, R., Parenti, G., Ballabio, A., and Raben, N. (2013) Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. EMBO Mol Med 5, Martel, R. R., Klicius, J., and Galet, S. (1977) Inhibition of the immune response by rapamycin, a new antifungal antibiotic. Can J Physiol Pharmacol 55, 48-51

19 Page Ye, L., Varamini, B., Lamming, D. W., Sabatini, D. M., and Baur, J. A. (2012) Rapamycin has a biphasic effect on insulin sensitivity in C2C12 myotubes due to sequential disruption of mtorc1 and mtorc2. Frontier Genet 3, Young, S. P., Zhang, H., Corzo, D., Thurberg, B. L., Bali, D., Kishnani, P. S., and Millington, D. S. (2009) Long-term monitoring of patients with infantile-onset Pompe disease on enzyme replacement therapy using a urinary glucose tetrasaccharide biomarker. Genet Med 11, Bjorkoy, G., Lamark, T., Pankiv, S., Overvatn, A., Brech, A., and Johansen, T. (2009) Monitoring autophagic degradation of p62/sqstm1. Methods Enzymol 452,

20 Page 20 Figure Legends Figure 1. Enhanced efficacy from clenbuterol treatment following AAV2/8-MHCK7hGAA administration. Vector-injected (2x10 11 vp/mouse) mice were treated with clenbuterol (2) following vector administration (n=12), vector alone (n=10), or untreated (n=8) and analyzed at 12 weeks. Statistically significant differences were demonstrated for clenbuterol treated mice, in comparison with mice treated with vector alone (one-way ANOVA, Tukey post test). (A) Introduced GAA is bound to CI-MPR to allow trafficking to the lysosomes, where it is activated and subsequently reduced lysosomal glycogen. (B) GAA activity. Normal GAA activity in wildtype mice shown for comparison. (C) Glycogen content. (D) Urinary biomarker (Glc 4 ) analyzed as described (37). *=p<0.05; **=p<0.01, ***=p< (E) Correlation of glycogen content in different tissues with urinary Glc 4. Figure 2. Effect of clenbuterol in vector-treated DKO mice. Vector-injected (2x10 10 vp/mouse) mice were treated with clenbuterol (2) following vector administration (n=6), or vector alone (n=5) and analyzed at 12 weeks. Statistically significant differences were demonstrated for clenbuterol treated mice, in comparison with mice treated with vector alone (paired t-test). (A) GAA activity, and (B) glycogen content, and (C) Realtime PCR quantification of vector DNA in the heart (n=4 in each group for the latter analysis).. *=p<0.05; **=p<0.01, ***=p< Figure 3. Enhanced muscle function following clenbuterol treatment with AAV2/8-MHCK7hGAA (2x10 10 vp) administration. Vector-injected GAA-KO mice were treated with clenbuterol (2) following vector administration (n=12), or vector alone (n=10); vector injected DKO mice were treated with clenbuterol following vector administration (n=6), or vector alone (n=5); and all mice were analzyed at 12 weeks. Statistically significant differences were demonstrated for clenbuterol treated mice, in comparison with mice treated with vector alone (paired t-test). (A) Rotarod latency, (B) wirehang latency.

21 Page 21 Figure 4. Clenbuterol treatment decreases the presence of LAMP-2+ muscle fibers. (A) Immunofluorescent detection of LAMP-2 in quadriceps shows decrease in expression with AAV + Clenbuterol treatment, high expression in untreated GAA-KO control mice, and low expression in wild-type controls. Scale = 50 um (B) One way ANOVA was performed using JMP Pro 10 software to determine statistical significance (p<0.05). Combination treatment of AAV + Clenbuterol significantly decreased the abundance of LAMP-2 in both GAA-KO and DKO mice compared to AAV only treated and untreated controls. (# = p<0.05 vs WT; * = p<0.05 vs No treatment; ** = p<0.05). Figure 5. GAA transduction increases autophagic flux in murine quadriceps. A.) Immunoblot of SQSTM1/p62 (lower band) (38), and autophagosomal marker LC3. B.) Densitometric analysis of p62 in the same mice. C.) Densitometric analysis of LC3-II. Results displayed as mean ± SD (n=4 per group). *=p<0.05; **=p<0.01. Figure 6: GAA transduction reduces AKT and mtor signalling in murine quadriceps. A.) Immunoblot of phosphorylated and total AKT. B.) Densitometric analysis of AKT phosphorylation in the same mice. Results displayed as mean + SEM. C.) Ratio of phosphorylated to total AKT. D.) Immunoblot of phosphorylated and total p70s6k. E.) Densitometric analysis of p70s6k phosphorylation in the same mice. F.) Ratio of phosphorylated to total p70s6k. Results displayed as mean + SEM. C.) Ratio of phosphorylated to total AKT. Results displayed as mean ± SD (n=4 per group; except for Wildtype n=2). *=p<0.05; **=p<0.01, ***=p<0.001.

22 Table: Effects of clenbuterol upon autophagy in muscle Autophagosome Number Autophagic Flux (p62) mtor Activity (p-p70s6k) AKT Activity (pakt) (LC3-II) No treatment Vector Vector + Clenbuterol

23

24

25

26

27

28