Advancements in AAV-mediated Gene Therapy for Pompe Disease.

Pompe disease (glycogen storage disease type II) is caused by mutations in acid α-glucosidase (GAA) resulting in lysosomal pathology and impairment of the muscular and cardio-pulmonary systems. Enzyme replacement therapy (ERT), the only approved therapy for Pompe disease, improves muscle function by reducing glycogen accumulation but this approach entails several limitations including a short drug half-life and an antibody response that results in reduced efficacy. To address these limitations, new treatments such as gene therapy are under development to increase the intrinsic ability of the affected cells to produce GAA. Key components to gene therapy strategies include the choice of vector, promoter, and the route of administration. The efficacy of gene therapy depends on the ability of the vector to drive gene expression in the target tissue and also on the recipient's immune tolerance to the transgene protein. In this review, we discuss the preclinical and clinical studies that are paving the way for the development of a gene therapy strategy for patients with early and late onset Pompe disease as well as some of the challenges for advancing gene therapy.

Autophagy in skeletal muscle: implications for Pompe disease.

Pompe disease is caused by an inherited deficiency of acid a-glucosidase (GAA), a lysosomal enzyme that catalyzes the breakdown of glycogen to glucose. In the absence of GAA, enlarged, glycogen-laden lysosomes accumulate in multiple tissues, although the major clinical manifestations are seen in cardiac and skeletal muscle. For many years, it was believed that the rupture of glycogen-filled lysosomes was the major cause of the profound muscle damage observed in patients with Pompe disease. Here, we present evidence that a failure of productive autophagy in muscle tissue contributes strongly to disease pathology in both patients with Pompe disease and GAA-knockout mice. In the GAA-knockout mouse model, progressive accumulation of autophagic vesicles is restricted to Type II-rich muscle fibers. Not only does this build-up of autophagosomes disrupt the contractile apparatus in the muscle fibers, it also interferes with enzyme replacement therapy by acting as a sink for the recombinant enzyme and preventing its efficient delivery to the lysosomes. Our data indicate that a re-examination of the presumed pathological mechanism in Pompe disease is necessary, and suggest that successful treatment of patients with Pompe disease will require consideration of the dramatic failure of autophagy that occurs in this disease.

Biochemical and pharmacological characterization of different recombinant acid alpha-glucosidase preparations evaluated for the treatment of Pompe disease.

Pompe disease results in the accumulation of lysosomal glycogen in multiple tissues due to a deficiency of acid alpha-glucosidase (GAA). Enzyme replacement therapy for Pompe disease was recently approved in Europe, the U.S., Canada, and Japan using a recombinant human GAA (Myozyme, alglucosidase alfa) produced in CHO cells (CHO-GAA). During the development of alglucosidase alfa, we examined the in vitro and in vivo properties of CHO cell-derived rhGAA, an rhGAA purified from the milk of transgenic rabbits, as well as an experimental version of rhGAA containing additional mannose-6-phosphate intended to facilitate muscle targeting. Biochemical analyses identified differences in rhGAA N-termini, glycosylation types and binding properties to several carbohydrate receptors. In a mouse model of Pompe disease, glycogen was more efficiently removed from the heart than from skeletal muscle for all enzymes, and overall, the CHO cell-derived rhGAA reduced glycogen to a greater extent than that observed with the other enzymes. The results of these preclinical studies, combined with biochemical characterization data for the three molecules described within, led to the selection of the CHO-GAA for clinical development and registration as the first approved therapy for Pompe disease.

Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence.

The large size of the multinucleated muscle fibers of skeletal muscle makes their examination for structural and pathological defects a challenge. Sections and single fibers are accessible to antibodies and other markers but imaging of such samples does not provide a three-dimensional view of the muscle. Regrettably, bundles of fibers cannot be stained or imaged easily. Two-photon microscopy techniques overcome these obstacles. Second harmonic generation (SHG) by myosin filaments and two-photon excited fluorescence (2PEF) of mitochondrial and lysosomal components provides detailed structural information on unstained tissue. Furthermore, the infrared exciting light can penetrate several layers of muscle fibers and the minimal processing is particularly valuable for fragile biopsies. Here we demonstrate the usefulness of SHG, combined with 2PEF, to reveal enlarged lysosomes and accumulations of non-contractile material in muscles from the mouse model for the lysosomal storage disorder Pompe disease (PD), and in biopsies from adult and infant PD patients. SHG and 2PEF also detect sarcomeric defects that may presage the loss of myofibrils in atrophying muscle and signify loss of elasticity. The combination of SHG and 2PEF should be useful in the analysis and diagnosis of a wide range of skeletal muscle pathologies.

Role of autophagy in the pathogenesis of Pompe disease.

In Pompe disease, a deficiency of lysosomal acid alpha-glucosidase, glycogen accumulates in multiple tissues, but clinical manifestations are mainly due to skeletal and cardiac muscle involvement. A major advance has been the development of enzyme replacement therapy (ERT), which recently became available for Pompe patients. Based on clinical and pre-clinical studies, the effective clearance of skeletal muscle glycogen appears to be more difficult than anticipated. Skeletal muscle destruction and resistance to therapy remain unsolved problems. We have found that the cellular pathology in Pompe disease spreads to affect both the endocytic and autophagic pathways, leading to excessive autophagic buildup in therapy resistant muscle fibers of knockout mice. Furthermore, the autophagic buildup had a profound effect on the trafficking and processing of the therapeutic enzyme along the endocytic pathway. These findings may explain why ERT often falls short of reversing the disease process, and point to new avenues for the development of pharmacological intervention.

Enzyme replacement therapy in the mouse model of Pompe disease.

Deficiency of acid alpha-glucosidase (GAA) results in widespread cellular deposition of lysosomal glycogen manifesting as myopathy and cardiomyopathy. When GAA-/- mice were treated with rhGAA (20 mg/kg/week for up to 5 months), skeletal muscle cells took up little enzyme compared to liver and heart. Glycogen reduction was less than 50%, and some fibers showed little or no glycogen clearance. A dose of 100 mg/kg/week resulted in approximately 75% glycogen clearance in skeletal muscle. The enzyme reduced cardiac glycogen to undetectable levels at either dose. Skeletal muscle fibers with residual glycogen showed immunoreactivity for LAMP-1/LAMP-2, indicating that undigested glycogen remained in proliferating lysosomes. Glycogen clearance was more pronounced in type 1 fibers, and histochemical analysis suggested an increased mannose-6-phosphate receptor immunoreactivity in these fibers. Differential transport of enzyme into lysosomes may explain the strikingly uneven pattern of glycogen removal. Autophagic vacuoles, a feature of both the mouse model and the human disease, persisted despite glycogen clearance. In some groups a modest glycogen reduction was accompanied by improved muscle strength. These studies suggest that enzyme replacement therapy, although at much higher doses than in other lysosomal diseases, has the potential to reverse cardiac pathology and to reduce the glycogen level in skeletal muscle.

Muscle as a putative producer of acid alpha-glucosidase for glycogenosis type II gene therapy.

Glycogenosis type II (GSD II) is a lysosomal disorder affecting skeletal and cardiac muscle. In the infantile form of the disease, patients display cardiac impairment, which is fatal before 2 years of life. Patients with juvenile or adult forms can present diaphragm involvement leading to respiratory failure. The enzymatic defect in GSD II results from mutations in the acid alpha-glucosidase (GAA) gene, which encodes a 76 kDa protein involved in intralysosomal glycogen hydrolysis. We previously reported the use of an adenovirus vector expressing GAA (AdGAA) for the transduction of myoblasts and myotubes cultures from GSD II patients. Transduced cells secreted GAA in the medium, and GAA was internalized by receptor-mediated capture, allowing glycogen hydrolysis in untransduced cells. In this study, using a GSD II mouse model, we evaluated the feasibility of GSD II gene therapy using muscle as a secretary organ. Adenovirus vector encoding AdGAA was injected in the gastrocnemius of neonates. We detected a strong expression of GAA in the injected muscle, secretion into plasma, and uptake by peripheral skeletal muscle and the heart. Moreover, glycogen content was decreased in these tissues. Electron microscopy demonstrated the disappearance of destruction foci, normally present in untreated mice. We thus demonstrate for the first time that muscle can be considered as a safe and easily accessible organ for GSD II gene therapy.

Conditional tissue-specific expression of the acid alpha-glucosidase (GAA) gene in the GAA knockout mice: implications for therapy.

Both enzyme replacement and gene therapy of lysosomal storage disorders rely on the receptor-mediated uptake of lysosomal enzymes secreted by cells, and for each lysosomal disorder it is necessary to select the correct cell type for recombinant enzyme production or for targeting gene therapy. For example, for the therapy of Pompe disease, a severe metabolic myopathy and cardiomyopathy caused by deficiency of acid alpha-glucosidase (GAA), skeletal muscle seems an obvious choice as a depot organ for local therapy and for the delivery of the recombinant enzyme into the systemic circulation. Using knockout mice with this disease and transgenes containing cDNA for the human enzyme under muscle or liver specific promoters controlled by tetracycline, we have demonstrated that the liver provided enzyme far more efficiently. The achievement of therapeutic levels with skeletal muscle transduction required the entire muscle mass to produce high levels of enzyme of which little found its way to the plasma, whereas liver, comprising <5% of body weight, secreted 100-fold more enzyme, all of which was in the active 110 kDa precursor form. Furthermore, using tetracycline regulation, we somatically induced human GAA in the knockout mice, and demonstrated that the skeletal and cardiac muscle pathology was completely reversible if the treatment was begun early.

Identification and characterization of a tissue-specific silencer element in the first intron of the human acid maltase gene.

Deficiency of acid maltase (acid alpha-glucosidase), a lysosomal enzyme that degrades glycogen, results in glycogenosis type II, an autosomal recessive disease whose manifestations and severity largely depend on the level of residual enzyme activity. Previous studies have established that there are transcriptional control elements in the first intron; in particular a silencer responsive to Hes-1 and YY1 has been identified in the human hepatoma line, HepG2. This region functions as an enhancer in human fibroblasts. Here we have localized a silencer active in fibroblasts to a nearby 25-bp element in intron 1. This element repressed thymidine kinase promoter activity by about 50% in both orientations in human fibroblasts. This silencer, as with the previous one, is tissue specific since constructs containing this region are inactive in HepG2 cells. Electrophoretic mobility shift assay revealed three proteins specifically binding to the element in fibroblasts, and site-directed mutagenesis analysis indicated that all the three proteins binding to the element contribute to the silencer function. The data may be helpful for designing therapy to increase the level of enzyme, particularly when, as in most adults with the disease, there is reduced production of structurally normal enzyme.

Surprises of genetic engineering: a possible model of polyglucosan body disease.

BACKGROUND: The authors previously reported the generation of a knockout mouse model of Pompe disease caused by the inherited deficiency of lysosomal acid alpha-glucosidase (GAA). The disorder in the knockout mice (GAA-/-) resembles the human disease closely, except that the clinical symptoms develop late relative to the lifespan of the animals. In an attempt to accelerate the course of the disease in the knockouts, the authors increased the level of cytoplasmic glycogen by overexpressing glycogen synthase (GSase) or GlutI glucose transporter. METHODS: GAA-/- mice were crossed to transgenic mice overexpressing GSase or GlutI in skeletal muscle. RESULTS: Both transgenics on a GAA knockout background (GS/GAA-/- and GlutI/GAA-/-) developed a severe muscle wasting disorder with an early age at onset. This finding, however, is not the major focus of the study. Unexpectedly, the mice bearing the GSase transgene, but not those bearing the GlutI transgene, accumulated structurally abnormal polysaccharide (polyglucosan) similar to that observed in patients with Lafora disease, glycogenosis type IV, and glycogenosis type VII. Ultrastructurally, the periodic acid-Schiff (PAS)-positive polysaccharide inclusions were composed of short, amorphous, irregular branching filaments indistinguishable from classic polyglucosan bodies. The authors show here that increased level of GSase in the presence of normal glycogen branching enzyme (GBE) activity leads to polyglucosan accumulation. The authors have further shown that inactivation of lysosomal acid alpha-glucosidase in the knockout mice does not contribute to the process of polyglucosan formation. CONCLUSIONS: An imbalance between GSase and GBE activities is proposed as the mechanism involved in the production of polyglucosan bodies. The authors may have inadvertently created a "muscle polyglucosan disease" by simulating the mechanism for polyglucosan formation.

Intercellular transfer of the virally derived precursor form of acid alpha-glucosidase corrects the enzyme deficiency in inherited cardioskeletal myopathy Pompe disease.

Pompe disease is a lethal cardioskeletal myopathy in infants and results from genetic deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). Genetic replacement of the cDNA for human GAA (hGAA) is one potential therapeutic approach. Three months after a single intramuscular injection of 10(8) plaque-forming units (PFU) of E1-deleted adenovirus encoding human GAA (Ad-hGAA), the activity in whole muscle lysates of immunodeficient mice is increased to 20 times the native level. Direct transduction of a target muscle, however, may not correct all deficient cells. Therefore, the amount of enzyme that can be transferred to deficient cells from virally transduced cells was studied. Fibroblasts from an affected patient were transduced with AdhGAA, washed, and plated on transwell culture dishes to serve as donors of recombinant enzyme. Deficient fibroblasts were plated as acceptor cells, and were separated from the donor monolayer by a 22-microm pore size filter. Enzymatic and Western analyses demonstrate secretion of the 110-kDa precursor form of hGAA from the donor cells into the culture medium. This recombinant, 110-kDa species reaches the acceptor cells, where it can be taken up by mannose 6-phosphate receptor-mediated endocytosis. It then trafficks to lysosomes, where Western analysis shows proteolytic processing to the 76- and 70-kDa lysosomal forms of the enzyme. Patient fibroblasts receiving recombinant hGAA by this transfer mechanism reach levels of enzyme activity that are comparable to normal human fibroblasts. Skeletal muscle cell cultures from an affected patient were also transduced with Ad-hGAA. Recombinant hGAA is identified in a lysosomal location in these muscle cells by immunocytochemistry, and enzyme activity is transferred to deficient skeletal muscle cells grown in coculture. Transfer of the precursor protein between muscle cells again occurs via mannose 6-phosphate receptors, as evidenced by competitive inhibition with 5 mM mannose 6-phosphate. In vivo studies in GAA-knockout mice demonstrate that hepatic transduction with adenovirus encoding either murine or human GAA can provide a depot of recombinant enzyme that is available to heart and skeletal muscle through this mechanism. Taken together, these data show that the mannose 6-phosphate receptor pathway provides a useful strategy for cell-to-cell distribution of virally derived recombinant GAA.

Transcriptional regulation of the human acid alpha-glucosidase gene. Identification of a repressor element and its transcription factors Hes-1 and YY1.

Acid alpha-glucosidase, the product of a housekeeping gene, is a lysosomal enzyme that degrades glycogen. A deficiency of this enzyme is responsible for a recessively inherited myopathy and cardiomyopathy, glycogenesis type II. We have previously demonstrated that the human acid alpha-glucosidase gene expression is regulated by a silencer within intron 1, which is located in the 5'-untranslated region. In this study, we have used deletion analysis, electrophoretic mobility shift assay, and footprint analysis to further localize the silencer to a 25-base pair element. The repressive effect on the TK promoter was about 50% in both orientations in expression plasmid, and two transcriptional factors were identified with antibodies binding specifically to the element. Mutagenesis and functional analyses of the element demonstrated that the mammalian homologue 1 of Drosophila hairy and Enhancer of split (Hes-1) binding to an E box (CACGCG) and global transcription factor-YY1 binding to its core site function as a transcriptional repressor. Furthermore, the overexpression of Hes-1 significantly enhanced the repressive effect of the silencer element. The data should be helpful in understanding the expression and regulation of the human acid alpha-glucosidase gene as well as other lysosomal enzyme genes.

Molecular scanning for mutations in the insulin receptor substrate-1 (IRS-1) gene in Mexican Americans with Type 2 diabetes mellitus.

BACKGROUND: Insulin receptor substrate-1 (IRS-1) is an endogenous substrate for the insulin receptor tyrosine kinase, which plays an important role in insulin signaling. Mutations in the IRS-1 gene are associated in some populations with obesity and Type 2 diabetes. METHODS: To determine whether variation in the IRS-1 gene contributes to genetic susceptibility to insulin resistance and Type 2 diabetes in Mexican Americans, the entire coding region of the IRS-1 gene was screened for variation in 31 unrelated subjects with Type 2 diabetes using single-stranded conformational polymorphism analysis (SSCP) and dideoxy sequence analysis. Variants encoding amino acid substitutions were genotyped in 27 unrelated nondiabetic Mexican Americans and in all family members of subjects containing these variants, and association analyses were performed. To trace the ancestral origins of the variants, Iberian Caucasians and Pima Indians were also genotyped. RESULTS: Eight single base changes were found: four silent polymorphisms and four missense mutations (Ala94Thr, Ala512Pro, Ser892Gly and Gly971Arg). Allele frequencies were 0.009, 0.017, 0.017 and 0.043, respectively. There were no significant associations of any of these variants with diabetes, glucose or insulin levels during an oral glucose tolerance test, or with body mass index (BMI) in Mexican American families except for a modest association between the Ala94Thr variant and decreased BMI (30.4 kg/m(2) vs 24.0 kg/m(2); p=0.035). None of these four missense mutations were detected in Pima Indians. In Iberian Caucasians, neither Ala94Thr nor Ser892Gly were detected, and Ala512Pro was detected in only 0/60 diabetic patients and 1/60 nondiabetic controls. Gly971Arg was relatively more common in Iberian Caucasians with 12/58 diabetic patients and 7/60 nondiabetic controls being heterozygous for this variant (p=0.21 for comparison between diabetic and nondiabetic subjects). CONCLUSIONS: Ala94Thr, Ala512Pro and Ser892Gly mutation are rare in the populations studied. Gly971Arg, is more common in Mexican Americans and Caucasians, but is not a major contributor to genetic susceptibility to Type 2 diabetes.

Correction of glycogen storage disease type II by enzyme replacement with a recombinant human acid maltase produced by over-expression in a CHO-DHFR(neg) cell line.

Inherited genetic deficiency of lysosomal acid alpha glucosidase or acid maltase (GAA) results in the autosomal recessive glycogen storage disease type II (GSD II). To investigate whether we could generate a functional recombinant human GAA (rhGAA) for enzyme replacement therapy, we subcloned the cDNAs for human GAA and mouse dihydrofolate reductase (DHFR) into DHFR(neg) Chinese hamster ovary cells and established a stable cotransformant that expressed rhGAA. We cultured the recombinant cells in media with progressively increasing concentrations of methotrexate and found that human GAA enzyme activity increased to over 2,000 IU per gram protein. Importantly, the human GAA enzyme activity correlated to equivalent amounts of human GAA protein by rocketimmunoelectrophoresis. We confirmed that the human GAA enzyme activity corresponded to an amplification in human GAA mRNA by Northern analysis and human GAA cDNA copy number by Southern analysis. Exposing the rhGAA to human GSDII fibroblast cells or patient's lymphocytes or monocytes resulted in uptake of the rhGAA and reversal of the enzymatic defect. Mannose-6-phosphate in the media blocked uptake. GAA -/- mice were treated with the rhGAA at 1 mg/kg, which resulted in heterozygous levels of GAA in tissues, most notably skeletal muscle, heart and diaphragm after two infusions. More importantly, after multiple infusions, hind, and fore-limb muscle weakness was reversed. This rhGAA would be ideal for enzyme replacement therapy in GSD II.

Conditional up-regulation of MHC class I in skeletal muscle leads to self-sustaining autoimmune myositis and myositis-specific autoantibodies.

In the human inflammatory myopathies (polymyositis and dermatomyositis), the early, widespread appearance of MHC class I on the surface of muscle cells and the occurrence of certain myositis-specific autoantibodies are striking features. We have used a controllable muscle-specific promoter system to up-regulate MHC class I in the skeletal muscles of young mice. These mice develop clinical, biochemical, histological, and immunological features very similar to human myositis. The disease is inflammatory, limited to skeletal muscles, self-sustaining, more severe in females, and often accompanied by autoantibodies, including, in some mice, autoantibodies to histidyl-tRNA synthetase, the most common specificity found in the spontaneous human disease, anti-Jo-1. This model suggests that an autoimmune disease may unfold in a highly specific pattern as the consequence of an apparently nonspecific event-the sustained up-regulation of MHC class I in a tissue-and that the specificity of the autoantibodies derives not from the specificity of the stimulus, but from the context, location, and probably the duration of the stimulus. This model further suggests that the presumed order of events as an autoimmune disease develops needs to be reconsidered.

Modulation of disease severity in mice with targeted disruption of the acid alpha-glucosidase gene.

Glycogen storage disease type II (GSDII) is a recessively inherited disorder caused by defects in lysosomal acid alpha-glucosidase. In an attempt to reproduce the range of clinical manifestations of the human illness we have created null alleles at the acid alpha-glucosidase locus (GAA) with several gene targeting strategies. In each knockout strain, enzyme activity was completely abolished and glycogen accumulated at indistinguishable rates. The phenotypes, however, differed strikingly. Acid alpha-glucosidase deficiency on a 129xC57BL/6 background resulted in a severe phenotype with progressive cardiomyopathy and profound muscle wasting similar to that in patients with glycogen storage disease type II. On a 129/C57BL/6xFVB background, homozygous mutants developed a milder phenotype with a later age of onset. Females were more affected than males irrespective of genetic background. As in humans with glycogen storage disease type II, therefore, other genetic loci affect the phenotypic expression of a single gene mutation.

Molecular scanning of the beta-3-adrenergic receptor gene in Pima Indians and Caucasians.

BACKGROUND: The beta-3-adrenergic receptor (beta3AR) stimulates lipolysis and thermogenesis in adipocytes. The Trp64Arg beta3AR variant is associated in some, but not all, studies with an earlier onset of Type 2 diabetes mellitus and features of the insulin resistance syndrome. Functional studies as to the role of the Trp64Arg variant have been inconclusive. Earlier studies screened the beta3AR gene in only ten obese, diabetic Pima Indians. Potentially another yet to be identified polymorphism in the beta3AR gene in linkage disequilibrium with the Trp64Arg polymorphism could explain the findings in the association and functional studies. METHODS: We scanned the beta3AR gene in 20 diabetic Pima subjects and 20 Caucasian subjects using single stranded conformational polymorphism (SSCP) analysis. Variants were sequenced using dideoxy sequence analysis and further characterized using allele specific oligonucleotide hybridization (ASO) and RNA template specific-polymerase chain reaction (RS-PCR) assays. RESULTS: We found a guanine to thymidine substitution in the first intron, 14 bases from the splice donor site in both groups. In virtually all subjects, only two haplotypes were detected, Trp64/g1856 and Arg64/t1856, indicating that the g1856t polymorphism is in linkage disequilibrium with the Trp64Arg polymorphism. The g1856t substitution introduces a new consensus splice donor site which, if used, would encode a truncated protein. RNA levels of the two beta3AR alleles were approximately equal in omental adipose tissue of heterozygotes. No aberrantly spliced beta3AR mRNA was detected, indicating that the new consensus splice donor site is not used in vivo. CONCLUSION: The g1856t polymorphism is in linkage disequilibrium with the Trp64Arg variant, but does not appear to have a functional role.

Costimulatory markers in muscle of patients with idiopathic inflammatory myopathies and in cultured muscle cells.

In an attempt to understand the mechanisms of cell injury in the inflammatory myopathies, we analyzed the expression of costimulatory molecules, CTLA4, CD28, CD86, CD40, and CD154 as well as HLA class I, HLA class II, and ICAM-I in normal muscle and in muscle biopsies from patients with polymyositis (PM) or dermatomyositis (DM). By immunohistochemical staining, DM and PM biopsies showed the presence of CTLA4, CD28, CD86, and CD40 on inflammatory cells. More strikingly, however, low levels of CTLA4 and CD28 were observed on muscle cells. The expression of CTLA4 and CD28 on nonlymphoid cells has not been previously reported. These unexpected findings were confirmed in cultured normal human myoblasts: various proinflammatory cytokines induced the expression of CTLA4 and CD28 on normal human muscle cells. The sequences of the cDNAs were found to be identical to the sequences for these molecules in T cells. The data suggest a novel complexity in the network of cellular interactions between the infiltrated immune cells and the muscle cells in which the normal relationship between infiltrating inflammatory cells and target tissue is under a previously unrecognized set of controls.

Systemic correction of the muscle disorder glycogen storage disease type II after hepatic targeting of a modified adenovirus vector encoding human acid-alpha-glucosidase.

This report demonstrates that a single intravenous administration of a gene therapy vector can potentially result in the correction of all affected muscles in a mouse model of a human genetic muscle disease. These results were achieved by capitalizing both on the positive attributes of modified adenovirus-based vectoring systems and receptor-mediated lysosomal targeting of enzymes. The muscle disease treated, glycogen storage disease type II, is a lysosomal storage disorder that manifests as a progressive myopathy, secondary to massive glycogen accumulations in the skeletal and/or cardiac muscles of affected individuals. We demonstrated that a single intravenous administration of a modified Ad vector encoding human acid alpha-glucosidase (GAA) resulted in efficient hepatic transduction and secretion of high levels of the precursor GAA proenzyme into the plasma of treated animals. Subsequently, systemic distribution and uptake of the proenzyme into the skeletal and cardiac muscles of the GAA-knockout mouse was confirmed. As a result, systemic decreases (and correction) of the glycogen accumulations in a variety of muscle tissues was demonstrated. This model can potentially be expanded to include the treatment of other lysosomal enzyme disorders. Lessons learned from systemic genetic therapy of muscle disorders also should have implications for other muscle diseases, such as the muscular dystrophies.

Novel mutations in African American patients with glycogen storage disease Type II. Mutations in brief no. 209. Online.

The infantile form of GSD II (an inherited deficiency of the lysosomal enzyme, acid alpha-glucosidase, Pompe disease) is a severe and invariably fatal disease characterized by a rapidly progressive generalized hypotonia, hepatomegaly, and cardiomegaly. We have recently demonstrated that African American patients share a common nonsense R854X mutation in exon 18 (Becker et al., 1998). Two other mutations, D645E and M519V, have been identified in individual African American patients (Hermans et al., 1993a; Huie et al., 1994a). We describe here three novel mutations in this population group: a missense W481R in exon 10, a deletion of a T1441 in exon 10, and a splicing defect at the 5' donor site of intron 8 (IVS g+la) . The splicing defect is shared by two unrelated patients and it is linked to intragenic polymorphic sites identical to those found in patients bearing the common R854X mutation.