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Glycogen debranching enzyme deficiency in glycogen storage disease type III (GSD III) results in excessive glycogen accumulation in multiple tissues, primarily the liver, heart, and skeletal muscle. We recently reported that an adeno-associated virus vector expressing a bacterial debranching enzyme (pullulanase) driven by the ubiquitous CMV enhancer/chicken β-actin (CB) promoter cleared glycogen in major affected tissues of infant GSD IIIa mice. In this study, we developed a potentially novel dual promoter consisting of a liver-specific promoter (LSP) and the CB promoter for gene therapy in adult GSD IIIa mice. Ten-week treatment with an adeno-associated virus vector containing the LSP-CB dual promoter in adult GSD IIIa mice significantly increased pullulanase expression and reduced glycogen contents in the liver, heart, and skeletal muscle, accompanied by the reversal of liver fibrosis, improved muscle function, and a significant decrease in plasma biomarkers alanine aminotransferase, aspartate aminotransferase, and creatine kinase. Compared with the CB promoter, the dual promoter effectively decreased pullulanase-induced cytotoxic T lymphocyte responses and enabled persistent therapeutic gene expression in adult GSD IIIa mice. Future studies are needed to determine the long-term durability of dual promoter–mediated expression of pullulanase in adult GSD IIIa mice and in large animal models.

Glycogen storage disease type III (GSD III) is an inherited disorder caused by a deficiency of glycogen debranching enzyme (GDE), which results in the accumulation of abnormal glycogen (limit dextrin) in the cytoplasm of liver, heart, and skeletal muscle cells. Currently, there is no curative treatment for this disease. Gene therapy with adeno-associated virus (AAV) provides an optimal treatment approach for monogenic diseases like GSD III. However, the 4.6 kb human GDE cDNA is too large to be packaged into a single AAV vector due to its small carrying capacity. To overcome this limitation, we tested a new gene therapy approach in GSD IIIa mice using an AAV vector ubiquitously expressing a smaller bacterial GDE, Pullulanase, whose cDNA is 2.2 kb. Intravenous injection of the AAV vector (AAV9-CB-Pull) into 2-week-old GSD IIIa mice blocked glycogen accumulation in both cardiac and skeletal muscles, but not in the liver, accompanied by the improvement of muscle functions. Subsequent treatment with a liver-restricted AAV vector (AAV8-LSP-Pull) reduced liver glycogen content by 75% and reversed hepatic fibrosis while maintaining the effect of AAV9-CB-Pull treatment on heart and skeletal muscle. Our results suggest that AAV-mediated gene therapy with Pullulanase is a possible treatment for GSD III.

Pompe disease, a severe and often fatal neuromuscular disorder, is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). The disease is characterized by the accumulation of excess glycogen in the heart, skeletal muscle, and CNS. Currently approved enzyme replacement therapy or experimental adeno-associated virus (AAV)-mediated gene therapy has little effect on CNS correction. Here we demonstrate that a newly developed AAV-PHP.B vector can robustly transduce both the CNS and skeletal muscles in GAA-knockout (GAAKO) mice. A single intravenous injection of an AAV-PHP.B vector expressing human GAA under the control of cytomegalovirus (CMV) enhancer-chicken β-actin (CB) promoter into 2-week-old GAAKO mice resulted in widespread GAA expression in the affected tissues. Glycogen contents were reduced to wild-type levels in the brain and heart, and they were significantly decreased in skeletal muscle by the AAV treatment. The histological assay showed no visible glycogen in any region of the brain and spinal cord of AAV-treated mice. In this study, we describe a set of behavioral tests that can detect early neurological deficits linked to extensive lysosomal glycogen accumulation in the CNS of untreated GAAKO mice. Furthermore, we demonstrate that the therapy can help prevent the development of these abnormalities. Keywords: Pompe disease, acid alpha-glucosidase deficiency, gene therapy, AAV-PHP.B vector, glycogen storage, CNS, neurological deficits

A major obstacle to enzyme replacement therapy (ERT) with recombinant human acid-α-glucosidase (rhGAA) for Pompe disease is the development of high titers of anti-rhGAA antibodies in a subset of patients, which often leads to a loss of treatment efficacy. In an effort to induce sustained immune tolerance to rhGAA, we supplemented the rhGAA therapy with a weekly intravenous injection of synthetic vaccine particles carrying rapamycin (SVP-Rapa) during the first 3 weeks of a 12-week course of ERT in GAA-KO mice, and compared this with three intraperitoneal injections of methotrexate (MTX) per week for the first 3 weeks. Empty nanoparticles (NP) were used as negative control for SVP-Rapa. Co-administration of SVP-Rapa with rhGAA resulted in more durable inhibition of anti-rhGAA antibody responses, higher efficacy in glycogen clearance in skeletal muscles, and greater improvement of motor function than mice treated with empty NP or MTX. Body weight loss was observed during the MTX-treatment but not SVP-Rapa-treatment. Our data suggest that co-administration of SVP-Rapa may be an innovative and safe strategy to induce durable immune tolerance to rhGAA during the ERT in patients with Pompe disease, leading to improved clinical outcomes.

Clear cell foci (CCF) of the liver are considered to be pre-neoplastic lesions of hepatocellular adenomas and carcinomas. They are hallmarked by glycogen overload and activation of AKT (v-akt murine thymoma viral oncogene homolog)/mTOR (mammalian target of rapamycin)-signaling. Here, we report the transcriptome and proteome of CCF extracted from human liver biopsies by laser capture microdissection. We found 14 genes and 22 proteins differentially expressed in CCF and the majority of these were expressed at lower levels in CCF. Using immunohistochemistry, the reduced expressions of STBD1 (starch-binding domain-containing protein 1), USP28 (ubiquitin-specific peptidase 28), monad/WDR92 (WD repeat domain 92), CYB5B (Cytochrome b5 type B), and HSPE1 (10 kDa heat shock protein, mitochondrial) were validated in CCF in independent specimens. Knockout of Stbd1, the gene coding for Starch-binding domain-containing protein 1, in mice did not have a significant effect on liver glycogen levels, indicating that additional factors are required for glycogen overload in CCF. Usp28 knockout mice did not show changes in glycogen storage in diethylnitrosamine-induced liver carcinoma, demonstrating that CCF are distinct from this type of cancer model, despite the decreased USP28 expression. Moreover, our data indicates that decreased USP28 expression is a novel factor contributing to the pre-neoplastic character of CCF. In summary, our work identifies several novel and unexpected candidates that are differentially expressed in CCF and that have functions in glycogen metabolism and tumorigenesis.

We report our recent calculations for the generalized parton distribution functions of the $\rho$ meson with the help of a light-front constituent quark model. The electromagnetic form factors and structure functions of the system are given. Moreover, we also show our results for its gravitational form factors (or energy-momentum tensor form factors) and for other mechanical properties, like its mass distributions, pressures, share-forces, and $D-$term.

Approximately 35–40% of patients with classic infantile Pompe disease treated with enzyme replacement therapy (ERT) develop high, sustained antibody titers against the therapeutic enzyme alglucosidase alfa, which abrogates the treatment efficacy. Induction of antigen-specific immune tolerance would greatly enhance ERT for these patients. Here we show that a short-course treatment with non-depleting anti-CD4 monoclonal antibody successfully induced long-term ERT-specific immune tolerance in Pompe disease mice. Our data suggest an effective adjuvant therapy to ERT.

SUMMARY Glycogen storage disease type IIIa (GSD IIIa) is an autosomal recessive disease caused by deficiency of glycogen debranching enzyme (GDE) in liver and muscle. The disorder is clinically heterogeneous and progressive, and there is no effective treatment. Previously, a naturally occurring dog model for this condition was identified in curly-coated retrievers (CCR). The affected dogs carry a frame-shift mutation in the GDE gene and have no detectable GDE activity in liver and muscle. We characterized in detail the disease expression and progression in eight dogs from age 2 to 16 months. Monthly blood biochemistry revealed elevated and gradually increasing serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) activities; serum creatine phosphokinase (CPK) activity exceeded normal range after 12 months. Analysis of tissue biopsy specimens at 4, 12 and 16 months revealed abnormally high glycogen contents in liver and muscle of all dogs. Fasting liver glycogen content increased from 4 months to 12 months, but dropped at 16 months possibly caused by extended fibrosis; muscle glycogen content continually increased with age. Light microscopy revealed significant glycogen accumulation in hepatocytes at all ages. Liver histology showed progressive, age-related fibrosis. In muscle, scattered cytoplasmic glycogen deposits were present in most cells at 4 months, but large, lake-like accumulation developed by 12 and 16 months. Disruption of the contractile apparatus and fraying of myofibrils was observed in muscle at 12 and 16 months by electron microscopy. In conclusion, the CCR dogs are an accurate model of GSD IIIa that will improve our understanding of the disease progression and allow opportunities to investigate treatment interventions.

The vector-mediated gene therapy with adeno-associated virus (AAV) is capable of inducing tolerance to introduced glucosidase (GAA) and enhancing the efficacy of enzyme replacement therapy (ERT) in cross-reacting immunologic material (CRIM)-negative patients with Pompe disease and in patients with other lysosomal storage diseases.

Pompe disease has resisted enzyme replacement therapy with acid ct-glucosidase (GAA), which has been attributed to inefficient cation-independent mannose-6-phosphate receptor (CI-MPR) mediated uptake. We evaluated 132-agonist drugs, which increased CI-MPR expression in GAA knockout (KO) mice. Clenbuterol along with a low-dose adeno-associated virus vector increased Rotarod latency by 75% at 4 wk, in comparison with vector alone (P<2x10-5). Glycogen content was lower in skeletal muscles, including soleus (P<0.01), extensor digitorum longus (EDL; P<0.001), and tibialis anterior (P<0.05) following combination therapy, in comparison with vector alone. Glycogen remained elevated in the muscles following clenbuterol alone, indicating an adjunctive effect with gene therapy. Elderly GAA-KO mice treated with combination therapy demonstrated 2-fold increased wire-hang latency, in comparison with vector or clenbuterol alone (P<0.001). The glycogen content of skeletal muscle decreased following combination therapy in elderly mice (P<0.05). Finally, CI-MPR-KO/GAA-KO mice did not respond to combination therapy, indicating that clenbuterol's effect depended on CI-MPR expression. In summary, adjunctive β2-agonist treatment increased CI-MPR expression and enhanced efficacy from gene therapy in Pompe disease, which has implications for other lysosomal storage disorders that involve primarily the brain. [ABSTRACT FROM AUTHOR]

Infantile Pompe disease progresses to a lethal cardiomyopathy in absence of effective treatment. Enzyme-replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) has been effective in most patients with Pompe disease, but efficacy was reduced by high-titer antibody responses. Immunomodulatory gene therapy with a low dose adeno-associated virus (AAV) vector (2 × 1010 particles) containing a liver-specific regulatory cassette significantly lowered immunoglobin G (IgG), IgG1, and IgE antibodies to GAA in Pompe disease mice, when compared with mock-treated mice (P < 0.05). AAV-LSPhGAApA had the same effect on GAA-antibody production whether it was given prior to, following, or simultaneously with the initial GAA injection. Mice given AAV-LSPhGAApA had significantly less decrease in body temperature (P < 0.001) and lower anaphylactic scores (P < 0.01) following the GAA challenge. Mouse mast cell protease-1 (MMCP-1) followed the pattern associated with hypersensitivity reactions (P < 0.05). Regulatory T cells (Treg) were demonstrated to play a role in the tolerance induced by gene therapy as depletion of Treg led to an increase in GAA-specific IgG (P < 0.001). Treg depleted mice were challenged with GAA and had significantly stronger allergic reactions than mice given gene therapy without subsequent Treg depletion (temperature: P < 0.01; symptoms: P < 0.05). Ubiquitous GAA expression failed to prevent antibody formation. Thus, immunomodulatory gene therapy could provide adjunctive therapy in lysosomal storage disorders treated by enzyme replacement. [ABSTRACT FROM AUTHOR]

Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) is an inherited muscular dystrophy caused by deficiency in the activity of the lysosomal enzyme acid α-glucosidase (GAA). We hypothesized that chimeric GAA containing an alternative signal peptide could increase the secretion of GAA from transduced cells and enhance the receptor-mediated uptake of GAA in striated muscle. The relative secretion of chimeric GAA from transfected 293 cells increased up to 26-fold. Receptor-mediated uptake of secreted, chimeric GAA corrected cultured GSD-II patient cells. High-level hGAA was sustained in the plasma of GSD-II mice for 24 weeks following administration of an AAV2/8 vector encoding chimeric GAA; furthermore, GAA activity was increased and glycogen content was significantly reduced in striated muscle and in the brain. Administration of only 1 × 1010 vector particles increased GAA activity in the heart and diaphragm for > 18 weeks, whereas 3 × 1010 vector particles increased GAA activity and reduced glycogen content in the heart, diaphragm, and quadriceps. Furthermore, an AAV2/2 vector encoding chimeric GAA produced secreted hGAA for > 12 weeks in the majority of treated GSD-II mice. Thus, chimeric, highly secreted GAA enhanced the efficacy of AAV vector-mediated gene therapy in GSD-II mice.Molecular Therapy (2006) 14, 822–830; doi: 10.1016/j.ymthe.2006.08.001 [ABSTRACT FROM AUTHOR]

We have previously demonstrated that activation of the Ras/Mapk pathways is required for transforming growth factor β (TGF-β) induction of TGF-β1 expression. Here we examined the role of the Ras/Mapk pathways in TGF-β induction of urokinase-type plasminogen activator receptor (uPAR) expression in untransformed intestinal epithelial cells (IECs). TGF-β activated the stress-activated protein kinases (Sapk)/c-Jun N-terminal kinases (JNKs) within 5–10 min, an effect that preceeded TGF-β induction of uPAR expression in these cells. TGF-β induction of both JNK1 activity and JunD phosphorylation was blocked by expression of a dominant-negative mutant of the type II TGF-β receptor (DN TβRII), a dominant-negative mutant of MKK4 (DN MKK4), or a dominant-negative mutant of Ras (RasN17), or by the addition of the JNK inhibitor SP600125. TGF-β also induced AP-1 complex formation at the distal AP-1 site (-184 to -178) of the uPAR promoter within 2 h of TGF-β addition, consistent with the time-dependent up-regulation of uPAR expression. The primary components present in the TGF-β-stimulated AP-1 complex bound to the uPAR promoter were Jun D and Fra-2. Moreover, addition of SP600125, or expression of DN MKK4 or DN TβRII, blocked TGF-β up-regulation of uPAR in IECs. Accordingly, our results indicate that TGF-β activates the Ras/MKK4/JNK1 signaling cascade, leading to induction of AP-1 activity, which, in turn, up-regulates uPAR expression. Our results also indicate that the type II TGF-β receptor (RII) is required for TGF-β activation of JNK1 and the resulting up-regulation of uPAR expression. J. Cell. Physiol. 199: 284–292, 2004© 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) is a genetic muscle disorder caused by a deficiency of acid-α-glucosidase (GAA; acid maltase; EC 3.2.1.20), which results in glycogen accumulation, primarily involving the heart and skeletal muscle. Previously, we provided evidence that an AAV2/6 vector containing a muscle-specific creatine kinase (MCK) promoter leads to persistent, high-level human GAA expression and glycogen clearance in the injected muscle in an immunocompetent GAA-KO mouse model. Muscle-restricted expression of hGAA provoked only a humoral (not cellular) immune response. We hypothesized that systemic administration of an AAV2/8 vector containing a MCK promoter could provide long-term efficacy in multiple muscles in GAA-KO mice. We intravenously administered either a low dose (1x1011 vg, n=3) or high dose (1x1012 vg, n=4) of the MCK-containing AAV2/8 vector in adult GAA-KO mice. At 18 weeks post-injection, GAA activity was significantly elevated only for the high-dose group to greater than the level for normal mice in heart (1.5-fold increase) and skeletal muscle (3.2-fold increase). The glycogen content was reduced by 46% and 50% in heart and quadriceps respectively, when compared to untreated, age-matched GAA-KO mice. Periodic acid-Schiff staining of glycogen confirmed correction of glycogen accumulation in individual myofibers of heart and quadriceps of AAV-treated mice. Low-level GAA activity was observed in liver and diaphragm, which is most likely related to lower activity of the MCK promoter in those tissues, because higher copy number of vector genome were present in liver (109.3 vg/cell) and diaphragm (1.63 vg/cell) than that in heart (0.47 vg/cell) and quadriceps (0.99 vg/cell) as determined by quantitative real-time PCR. In order to characterize transgene expression in different types of muscle fibers, an AAV vector encoding GFP under control of the MCK promoter was administered intravenously (1x1012 vg). Expression of GFP was detected in heart, quadriceps and soleus muscles, but not in EDL (extensor digitorum longus) muscle at 18 weeks after vector injection. Fiber-typing confirmed that soleus muscle consists mainly of type I (slow) myofibers, while EDL consists of type IIb (fast) myofiber in GAA-KO mice. In summary, persistent correction of glycogen storage in type I fibers of the heart and skeletal muscle was achieved with an AAV2/8 vector containing a MCK promoter in GAA-KO mice. This approach could be considered for muscle-targeted gene therapy in Pompe disease.Molecular Therapy (2006) 13, S217–S217; doi: 10.1016/j.ymthe.2006.08.636 [ABSTRACT FROM AUTHOR]

Glycogen storage disease type Ia (GSD-Ia) profoundly impairs glucose release by the liver due to glucose-6-phosphatase (G6Pase) deficiency. An adeno-associated virus (AAV) containing a small human G6Pase transgene was pseudotyped with AAV8 (AAV2/8) to optimize liver tropism. Survival was prolonged in 2-week-old G6Pase (–/–) mice by 600-fold fewer AAV2/8 vector particles (vp), in comparison to previous experiments involving this model (2 × 109 vp; 3 × 1011 vp/kg). When the vector was pseudotyped with AAV1, survival was prolonged only at a higher dose (3 × 1013 vp/kg). The AAV2/8 vector uniquely prevented hypoglycemia during fasting and fully corrected liver G6Pase deficiency in GSD-Ia mice and dogs. The AAV2/8 vector has prolonged survival in three GSD-Ia dogs to >11 months, which validated this strategy in the large animal model for GSD-Ia. Urinary biomarkers, including lactate and 3-hydroxybutyrate, were corrected by G6Pase expression solely in the liver. Glycogen accumulation in the liver was reduced almost to the normal level in vector-treated GSD-Ia mice and dogs, as was the hepatocyte growth factor (HGF) in GSD-Ia mice. These preclinical data demonstrated the efficacy of correcting hepatic G6Pase deficiency, and support the further preclinical development of AAV vector–mediated gene therapy for GSD-Ia.Molecular Therapy (2008); 16 4 665–672 doi:10.1038/mt.2008.15 [ABSTRACT FROM AUTHOR]

A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but why and how glycogen is transported to the lysosomes are unclear. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knockout mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knockout mice. In fasted double knockout mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at age of 3 months and by 60% at 13 months compared with GAA knockout mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in dKO mice restored the liver lysosomal glycogen content to the level of GAA knockout mice, so did a mutant lacking the Atg8 family interacting motif (AIM), and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal CBM20 starch binding domain interlinked by an HA-tag. Our results demonstrate that Stbd1 plays a dominate role in glycogen transport to lysosomes in liver and the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. [ABSTRACT FROM AUTHOR]

The glucose-6-phosphatase (G6Pase) deficiency of GSD-Ia prevents the conversion of glycogen to glucose in the liver. G6Pase overexpression was associated with blood glucose elevations; therefore, unregulated G6Pase expression should be avoided when considering strategies for gene therapy for GSD-Ia. A minimal G6Pase promoter containing insulin-responsive sequence elements was placed upstream of the human G6Pase cDNA in AAV-GRPhG6PpA. This vector was pseudotyped as AAV2/8 and administered to two week-old affected GSD-Ia mice (3 × 1011 DNase-resistant vector particles; 6 × 1013 particles/kg). An AAV2/8 vector containing the universally active CB promoter, AAV-CBcG6PpA, was also administered to affected GSD-Ia mice; however, the CB promoter is NOT glucose-responsive. Importantly, G6Pase expression with the glucose-responsive promoter (GRP) was as effective as the CB promoter/enhancer at correcting hypoglycemia. AAV-GRPhG6PpA significantly corrected hypoglycemia in fasting, affected GSD-Ia mice at 4 weeks of age. Pseudotyping AAV-GRPhG6PpA as AAV2/8 or AAV2/1 corrected hypoglycemia equivalently in GSD-Ia mice. Resolution of hypoglycemia was associated with the correction of other biochemical abnormalities, including hypercholesterolemia and G6Pase deficiency/glycogen storage in the liver. Furthermore, AAV- GRPhG6pA promoted normal growth and prolonged the survival of GSD-Ia mice by greater than 6 months, in comparison to untreated, affected mice which failed to grow and did not survive weaning. AAV-GRPhG6PpA was modified by deleting the D sequence from the terminal repeat to package scAAV-GRPhG6PpA, thereby increasing the efficiency of liver transduction. When scAAV-GRPhG6PpA, pseudotyped as AAV2/8, was administered to 2 week- old affected G6Pase-KO mice (3 × 1011 vector particles), blood glucose was significantly increased (90 +/− 16 mg/dl versus 31 +/− 19 mg/dL for untreated, affected mice; p<0.005). Furthermore, the number of scAAV vector particles administered could be reduced 10-fold compared to the analogous non-scAAV vector, while still correcting hypoglycemia and prolonging survival. An AAV vector encoding a liver-specific promoter (AAV-LSPhG6PpA) to drive unregulated G6Pase expression was administered to unaffected (wild-type) G6Pase-KO mice at 2 weeks of age and a glucose tolerance test was performed at 1 month old. The glucose tolerance test detected elevated glucose, in comparison to untreated wild-type mice, which reflected glucose intolerance related to unregulated G6Pase expression driven by the LSP. In contrast, AAV-GRPhG6PpA administration did not cause glucose intolerance in affected or wild-type mice. Hence, an scAAV vector efficaciously delivered a regulated transgene to liver without causing glucose intolerance in GSD-Ia.Molecular Therapy (2006) 13, S189–S189; doi: 10.1016/j.ymthe.2006.08.557 [ABSTRACT FROM AUTHOR]