Identification of a missense mutation in an adult-onset patient with glycogenosis type II expressing only one allele.

The lysosomal enzyme acid alpha glucosidase (GAA) or acid maltase is deficient in glycogen storage disease type II. We sought to determine the molecular basis for the disease in an adult-onset patient, unusual for very low enzyme activity similar to that seen with the infantile-onset form and with a previously reported defect in phosphorylation. We constructed cDNA and genomic DNA libraries from the patient's cell line (GM 1935) and determined the nucleotide sequence of the coding region. There were three base-pair substitutions in one allele (C1935 to A; G2446 to A and C2780 to T), all predicting amino acid changes (Asp-645 to Glu; Val-816 to Ile and Thr-927 to Ile). To determine which of the three base-pair substitutions resulted in loss of enzyme activity, we next utilized primer-directed mutagenesis and transient gene expression in an SV40-immortalized GAA-deficient fibroblast cell line. Only the construct containing the G2446 to A mutation (Val-816 to Ile) lost GAA enzyme activity, while the other two substitutions (including the Thr-927 to Ile change that predicts a loss of a potential site for N-linked glycosylation and mannose phosphorylation) each resulted in enzyme activity equal to the control. Analysis of RFLPs in genomic DNA, as well as sequence analysis for the three base-pair alterations, indicated that the patient was a genetic compound. We next digested PCR-amplified cDNA (reverse-transcribed from RNA) with Aat II to detect the base-pair 1935 substitution and found that virtually all of the mRNA was derived from the allele with the three base-pair substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of Tolerance to a Recombinant Human Enzyme,Acid Alpha-Glucosidase,in Enzyme Deficient Knockout Mice

When knockout mice are used to test the efficacy of recombinant human proteins, the animals often develop antibodies to the enzyme, precluding long-term pre-clinical studies. This has been a problem with a number of models, for example, the evaluation of gene or enzyme replacement therapies in a knockout model of glycogen storage disease type II (GSDII; Pompe syndrome). In this disease, the lack of acid alpha-glucosidase (GAA) results in lysosomal accumulation of glycogen, particularly in skeletal and cardiac muscle. Here, we report that in a GAA-deficient mouse model of GSDII, low levels of transgene-encoded human GAA expressed in skeletal muscle or liver dramatically blunt or abolish the immune response to human recombinant protein. Of two low expression transgenic lines, only the liver-expressing line exhibited a profound GAA deficiency in skeletal muscle and heart indistinguishable from that in the original knockouts. The study suggests that the induction of tolerance in animal models of protein deficiencies could be achieved by restricting the expression of a gene of interest to a particular, carefully chosen tissue.     

Identification of the base-pair substitution responsible for a human acid alpha glucosidase allele with lower "affinity" for glycogen (GAA 2) and transient gene expression in deficient cells

The lysosomal enzyme termed acid alpha glucosidase (GAA), or acid maltase, is genetically polymorphic, with three alleles segregating in the normal population. The rarer GAA 2 allozyme has a lower affinity for glycogen and starch but not for lower-molecular-weight substrates. The GAA 2 allozyme can be detected by "affinity" electrophoresis in starch gel, since the lower affinity for the starch matrix results in a more rapid migration to the anode. Previously, we have isolated and sequenced the cDNA for GAA and transiently expressed the cDNA in deficient fibroblasts. In order to determine the molecular basis for the GAA 2 allozyme, we constructed a cDNA and a genomic DNA library from a GAA 2 cell line and determined the nucleotide sequence of the coding region. Only a single base-pair substitution of an A for a G at base-pair 271 was found, resulting in substitution of asparagine for aspartic acid at codon 91. This amino acid substitution is consistent with the more basic pI of the GAA 2 enzyme. The base-pair substitution also abolishes a Taq-I site, predicting the generation of a larger DNA fragment. This larger Taq-I fragment was also seen in two other individuals expressing the GAA 2 allozyme. A 5' fragment containing the base-pair substitution was ligated to the remaining 3' cDNA from a GAA 1 allele and cloned into an expression vector, and the hybrid cDNA was transiently expressed in SV40-transformed GAA-deficient fibroblasts. The enzyme activity exhibited the altered mobility of the GAA 2 allozyme, as demonstrated by electrophoresis in starch gel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a missense mutation in one allele of a patient with Pompe disease, and use of endonuclease digestion of PCR-amplified RNA to demonstrate lack of mRNA expression from the second allele.

Infantile-onset glycogen storage disease type II, or Pompe disease, results from a genetic deficiency of the lysosomal enzyme acid alpha glucosidase (GAA). Sequencing of the cDNA from a cell line (GM 244) derived from a patient with Pompe disease demonstrated a T953-to-C transition that predicted a methionine-to-threonine substitution at codon 318. The basepair substitution resulted in loss of restriction-endonuclease sites for NcoI and StyI. Analysis of genomic DNA revealed both a normal and an abnormal NcoI fragment, indicating that the patient was a genetic compound. NcoI and StyI digestion of cDNA, amplified by PCR from reverse-transcribed RNA, demonstrated that greater than 95% of the GAA mRNA in GM 244 was derived from the allele carrying the missense mutation. The missense mutation was uncommon, since it was not detected in 37 additional GAA-deficient chromosomes, as determined by digestion of genomic DNA with NcoI and hybridization. The amino acid substitution predicts a new potential site for N-linked glycosylation, as well as major changes in secondary structure of the protein. We could confirm that the mutation was responsible for the enzyme deficiency by demonstrating that a hybrid minigene containing the mutation did not express GAA enzyme activity after transient gene expression. We have therefore now provided the first identification of a single-basepair missense mutation in a patient with Pompe disease and furthermore have demonstrated that the patient is a genetic compound with the second allele barely expressing mRNA.     

Identification of the promoter region and gene expression for human acid alpha glucosidase.

Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II. A cDNA containing the complete coding region was constructed and cloned into the expression vector pSV2 and was transiently transfected into an SV40 immortalized GAA deficient human fibroblast cell line which has undetectable levels of GAA enzyme activity and does not express GAA mRNA. Transfected cells had 4.9% of normal human fibroblast enzyme activity. Additionally a 5' 1.8 kb genomic fragment was ligated to the 5' end of the GAA cDNA construct and cloned into pUC19. Transient and stable transfection also resulted in expressed GAA enzyme activity in deficient fibroblast cells, indicating that the genomic fragment has GAA promoter function.     

Helios gene gun particle delivery for therapy of acid maltase deficiency

Autosomal recessive deficiency of lysosomal acid maltase (GAA) or glycogen storage disease type II (GSDII) results in a spectrum of phenotypes including a rapidly fatal infantile disorder (Pompe's), juvenile, and a late-onset adult myopathy. The infantile onset form presents as hypotonia with massive accumulation of glycogen in skeletal and heart muscle, with death due to cardiorespiratory failure. Adult patients with the slowly progressive form develop severe skeletal muscle weakness and respiratory failure. Particle bombardment is a safe, efficient physical method in which high-density, subcellular-sized particles are accelerated to high velocity to carry DNA into cells. Because it does not depend on a specific ligand, receptor, or biochemical features on cell surfaces, particle-mediated gene transfer can be readily applied to a variety of systems. We evaluated particle bombardment as a delivery system for therapy of GSDII. We utilized a vector carrying the CMV promoter linked to the human GAA cDNA. Human GSDII cell lines (fibroblasts and lymphoid) as well as ex vivo with adult-onset peripheral blood cells (lymphocytes and monocytes) were transiently transfected by bombardment with a Helios gene gun delivering gold particles coated with the GAA expression plasmid. All cell types showed an increase in human GAA activity greater than 50% of normal activity. Subsequently, GAA -/- mice were treated every 2 weeks for 4 months by particle bombardment to the epidermis of the lower back and hind limbs. Muscle weakness in the hind and forelimbs was reversed. These data suggest that particle delivery of the GAA cDNA by the Helios gene gun may be a safe, effective treatment for GSDII.     

The role of immune tolerance induction in restoration of the efficacy of ERT in Pompe disease

Pompe disease is a lysosomal storage disorder caused by deficiency in the enzyme acid α-glucosidase (GAA). Pompe disease is characterized by the accumulation of glycogen, predominantly in muscle tissue, leading to progressive muscle weakness, loss of motor, respiratory, and, in the infantile-onset form, cardiac function. Disease progression is highly variable depending on phenotype, but premature death due to respiratory complications occurs in most patients. Beginning in 2006, approved alglucosidase alfa enzyme replacement therapies [recombinant human (rh) GAA] have been available to treat Pompe patients. Treatment of classic infantile-onset patients, who manifest the severest form of the disease, with alglucosidase alfa (Myozyme?) has led to extended survival and an evolving understanding of the pathophysiology and course of the disease. Moreover, such treatment has brought to light the role of the immune response in abrogating the efficacy of rhGAA in classic infantile-onset patients with severe genetic mutations. Thus, optimization of treatment for such patients includes development and utilization of strategies to prevent or eliminate immune responses, including modulating the immune system (prophylactic and therapeutic immune tolerance induction regimens) and engineering the enzyme to be less immunogenic and more effective. Future research is also critical for evaluating and mitigating novel disease-associated pathologies uncovered by prolonged survival of infantile-onset patients including development of novel therapeutics, and for protein design strategies to increase delivery of enzyme replacement therapy to critical target tissues. Such efforts would be greatly bolstered by further development of predictive animal models and biomarkers to facilitate clinical trials and patient management. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

The genotype-phenotype correlation in Pompe disease

Pompe disease is an autosomal recessive lysosomal glycogen storage disorder that is caused by acid α-glucosidase (GAA) deficiency and is due to pathogenic sequence variations in the corresponding GAA gene. The correlation between genotypes and phenotypes is strict, in that patients with the most severe phenotype, classic infantile Pompe disease, have two pathogenic mutations, one in each GAA allele, that prevent the formation of GAA or totally obliterates its function. All patients with less progressive phenotypes have at least one sequence variation that allows normal or low level synthesis of GAA leading to the formation of analytically measurable, low level GAA activity in most cases. There is an overall trend of finding higher GAA enzyme levels in patients with onset of symptoms in adulthood when compared to patients who show clinical manifestations in early childhood, aged 0-5 years, with a rapidly progressive course, but who lack the severe characteristics of classic infantile Pompe disease. However, several cases have been reported of adult-onset disease with very low GAA activity, which in all those cases corresponds with the GAA genotype. The clinical diversity observed within a large group of patients with functionally the same GAA genotype and the same c.-32-13C?>?T haplotype demonstrates that modifying factors can have a substantial effect on the clinical course of Pompe disease, disturbing the GAA genotype-phenotype correlation. The present day challenge is to identify these factors and explore them as therapeutic targets.     

Novel GAA sequence variant c.1211 A > G reduces enzyme activity but not protein expression in infantile and adult onset Pompe disease

Pompe disease is a clinically and genetically heterogeneous autosomal recessive disorder caused by lysosomal acid α-glucosidase (GAA) deficiency. We report on two affected members of a non-consanguineous Caucasian family, including a classical infantile-onset patient with severe cardiomyopathy (IO) and his paternal grandmother with the adult-onset (AO) form. Two compound heterozygous sequence variants of the GAA gene were identified in each patient by mutation analyses (IO = c.1211A > G and c.1798C > T; AO = c.1211A > G and c.692 + 5G > T). For this study, the biochemical phenotype resulting from the missense mutation c.1211A > G in exon 8, which converts a highly conserved aspartate to glycine (p.Asp404Gly), was of specific interest because it had not been reported previously. Western blotting revealed a robust expression of all GAA isoforms in quadriceps muscle of both patients (fully CRIM positive), while enzymatic activity was 3.6% (IO) and 6.6% (AO) of normal controls. To further validate these findings, the c.1211A > G sequence variant was introduced in wild type GAA cDNA and over-expressed in HEK293T cells. Site-directed mutagenesis analyses confirmed that the mutation does not affect processing or expression of GAA protein, but rather impairs enzyme function. Similar results were reported for c.1798C > T (p.Arg600Cys), which further supports the biochemical phenotype observed in IO. The third mutation (c.692 + 5G > T, in intron 3) was predicted to affect normal splicing of the GAA mRNA, and qPCR indeed verified a 4-fold lower mRNA expression in AO. It is concluded that the novel sequence variant c.1211A > G results in full CRIM but significantly lower GAA activity, which in combination with c.1798C > T leads to infantile-onset Pompe disease. We surmise that the difference in disease severity between the two family members in this study is due to a milder effect of the intronic mutation c.692 + 5G > T (vs. c.1798C > T) on phenotype, partially preserving GAA activity and delaying onset in the proband (paternal grandmother).     

Correction of glycogenosis type 2 by muscle-specific lentiviral vector

Glycogen storage disease type II (GSDII) or Pompe disease is an inherited disease of glycogen metabolism caused by a lack of functional lysosomal acid α-glucosidase (GAA). Affected individuals store glycogen in lysosomes resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe form. We investigated for the first time the use of lentiviral vectors to correct the GSDII phenotype in human and murine GAA-deficient cells. Fibroblasts from infantile and adult GSDII patients were efficiently transduced by a GAA-expressing lentiviral vector placed under the control of the strong MND promoter, leading to a complete restoration of enzymatic activity. We also developed a muscle-specific lentiviral vector based on the synthetic C5–12 promoter and tested it on deficient myogenic satellite cells derived from a GSDII mouse model. GAA was expressed as a correctly processed protein allowing a complete enzymatic and metabolic correction in myoblasts and differentiated myotubes, as well as a significant mannose-6-phosphate (M6P)-dependent secretion reuptake by naive cells. Transduced cells showed lysosomal glycogen clearance, as demonstrated by electron microscopy. These results form the basis for a therapeutic approach of GSDII using lentiviral vector-mediated gene transfer into muscle stem cells.     

Genetic expression in partial adenosine deaminase deficiency. mRNA levels and protein turnover for the enzyme variants in human B-lymphoblast cell lines

A severe genetic deficiency of adenosine deaminase is causally associated with an autosomal recessive form of severe combined immunodeficiency disease, while subjects with absent erythrocyte but partial lymphocyte enzyme activity remain immunocompetent. The genetic expression of adenosine deaminase in B-lymphoblast cell lines derived from four unrelated subjects with the "partial" enzyme deficiency was examined. Enzymatic activity among these cell lines ranged from 5 to 50% of normal with the level of immunoreactive adenosine deaminase protein either proportional to enzyme activity or elevated in two of the cases. Northern blot analysis using a cDNA probe showed that adenosine deaminase mRNA in each of these cell lines was of normal expected size (1.6-1.8 kilobases) and was present in normal to above normal amounts. Rates of enzyme synthesis varied from 165 to 15% of normal. Adenosine deaminase protein degradation rates in these cell lines were 1.5 to almost 3 times faster than normal, consistent with the observed absence of the enzyme in erythrocytes. From these analyses apparent abnormalities in mRNA regulation, translation, and protein degradation can be identified among the partially adenosine deaminase-deficient cell lines studied. Ultimately, it will be essential to determine the nature of the protein mutation and the gene defect to define the structural alterations and functional abnormalities of enzyme variants isolated from subjects with partial adenosine deaminase deficiency.     

Enhanced response to enzyme replacement therapy in Pompe disease after the induction of immune tolerance

Pompe disease, which results from mutations in the gene encoding the glycogen-degrading lysosomal enzyme acid alpha -glucosidase (GAA) (also called "acid maltase"), causes death in early childhood related to glycogen accumulation in striated muscle and an accompanying infantile-onset cardiomyopathy. The efficacy of enzyme replacement therapy (ERT) with recombinant human GAA was demonstrated during clinical trials that prolonged subjects' overall survival, prolonged ventilator-free survival, and also improved cardiomyopathy, which led to broad-label approval by the U.S. Food and Drug Administration. Patients who lack any residual GAA expression and are deemed negative for cross-reacting immunologic material (CRIM) have a poor response to ERT. We previously showed that gene therapy with an adeno-associated virus (AAV) vector containing a liver-specific promoter elevated the GAA activity in plasma and prevented anti-GAA antibody formation in immunocompetent GAA-knockout mice for 18 wk, predicting that liver-specific expression of human GAA with the AAV vector would induce immune tolerance and enhance the efficacy of ERT. In this study, a very low number of AAV vector particles was administered before initiation of ERT, to prevent the antibody response in GAA-knockout mice. A robust antibody response was provoked in naive GAA-knockout mice by 6 wk after a challenge with human GAA and Freund's adjuvant; in contrast, administration of the AAV vector before the GAA challenge prevented the antibody response. Most compellingly, the antibody response was prevented by AAV vector administration during the 12 wk of ERT, and the efficacy of ERT was thereby enhanced. Thus, AAV vector-mediated gene therapy induced a tolerance to introduced GAA, and this strategy could enhance the efficacy of ERT in CRIM-negative patients with Pompe disease and in patients with other lysosomal storage diseases.     

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.     

Rescue of enzyme deficiency in embryonic diaphragm in a mouse model of metabolic myopathy: Pompe disease

Several human genetic diseases that affect striated muscle have been modeled by creating knockout mouse strains. However, many of these are perinatal lethal mutations that result in death from respiratory distress within hours after birth. As the diaphragm muscle does not contract until birth, the sudden increase in diaphragm activity creates permanent injury to the muscle causing it to fail to meet respiratory demands. Therefore, the impact of these mutations remains hidden throughout embryonic development and early death prevents investigators from performing detailed studies of other striated muscle groups past the neonatal stage. Glycogen storage disease type II (GSDII), caused by a deficiency in acid alpha-glucosidase (GAA), leads to lysosomal accumulation of glycogen in all cell types and abnormal myofibrillogenesis in striated muscle. Contractile function of the diaphragm muscle is severely affected in both infantile-onset and late-onset individuals, with death often resulting from respiratory failure. The knockout mouse model of GSDII survives well into adulthood despite the gradual weakening of all striated muscle groups. Using this model, we investigated the delivery of recombinant adeno-associated virus (rAAV) vectors encoding the human GAA cDNA to the developing embryo. Results indicate specific high-level transduction of diaphragm tissue, leading to activity levels up to 10-fold higher than normal and restoration of normal contractile function. Up to an estimated 50 vector copies per diploid genome were quantified in treated diaphragms. Histological glycogen staining of treated diaphragms revealed prevention of lysosomal glycogen accumulation in almost all fibers when compared with untreated controls. This method could be employed with disease models where specific rescue of the diaphragm would allow for increased survival and thus further investigation into the impact of the gene deletion on other striated muscle groups.     

Hyaluronidase increases the biodistribution of acid alpha-1,4 glucosidase in the muscle of Pompe disease mice: an approach to enhance the efficacy of enzyme replacement therapy

Pompe disease (glycogen storage disease type II) is a glycogen storage disease caused by a deficiency of the lysosomal enzyme, acid maltase/acid alpha-1,4 glucosidase (GAA). Deficiency of the enzyme leads primarily to intra-lysosomal glycogen accumulation, primarily in cardiac and skeletal muscles, due to the inability of converting glycogen into glucose. Enzyme replacement therapy (ERT) has been applied to replace the deficient enzyme and to restore the lost function. However, enhancing the enzyme activity to the muscle following ERT is relatively insufficient. In order to enhance GAA activity into the muscle in Pompe disease, efficacy of hyaluronidase (hyase) was examined in the heart, quadriceps, diaphragm, kidney, and brain of mouse model of Pompe disease. Administration of hyase 3000 U/mouse (intravenous) i.v. or i.p. (intraperitoneal) and 10 min later recombinant human GAA (rhGAA) 20 mg/kg i.v. showed more GAA activity in hyase i.p. injected mice compared to those mice injected with hyase via i.v. Injection of low dose of hyase (3000 U/mouse) or high dose of hyase (10,000 U/mouse) i.p. and 20 min or 60 min later 20 mg/kg rhGAA i.v. increased GAA activity into the heart, diaphragm, kidney, and quadriceps compared to hyase untreated mice. These studies suggest that hyase enhances penetration of enzyme into the tissues including muscle during ERT and therefore hyase pretreatment may be important in treating Pompe disease.     

Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10–12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.Subject terms: Cancer metabolism, CNS cancer     

Acid alpha-glucosidase deficiency: Identification and expression of a missense mutation (S529V) in a Japanese adult phenotype

We report a missense mutation in an adult Japanese patient with acid alpha-glucosidase (GAA) deficiency. A TC to GT transition at nucleotides 1585–1586, was identified. This transition resulted in an amino acid substitution of Ser-529 to Val (S529V) in exon 11. We also have demonstrated that the S529V mutation abolishes the catalytic activity of the enzyme. Our data suggest that this mutation is the cause of the clinical manifestation known as adult-onset GAA deficiency. The missense mutation described here is a new mutation, and the first identified in Japanese patients with GAA deficiency. Received: 23 May 1995     

Inverse relationship of skeletal muscle glycogen from wild-type and genetically modified mice to their phosphorylase a activity.

Leg muscle was biopsied and frozen for storage at -70 degrees C. from 5 wild-type mice, two knocked out acid alpha-glucosidase (GAA) gene mice, and seven glycogen synthase plus glucose muscle transporter transgenic mice. All of the wild-type mice had very little muscle glycogen (3.58 +/- 1.67 micromols glucosyl subunits per g muscle), and 52% or more of its glycogen phosphorylase activity without AMP (69% +/- 17% glycogen phosphorylase a). In contrast the GAA knockout and transgenic mice had glycogen ranging from 63 to 297 micromols glucosyl subunits per g muscle, and very little or no glycogen phosphorylase activity without 1.00 mM AMP (4.8% and less glycogen phosphorylase a). This suggests that there is an inverse relationship between mouse muscle phosphorylase a and the muscle's glycogen content.     

Characterization of the mutant N-acetylglucosaminylphosphotransferase in I-cell disease and pseudo-Hurler polydystrophy: complementation analysis and kinetic studies

Complementation was examined among various types of I-cell disease and pseudo-Hurler polydystrophy by monitoring N-acetylglucosaminylphosphotransferase activity in multinucleated cells produced by fusing pair combinations of cultured skin fibroblasts. Patients with the classical forms of these disorders (5 I-cell disease and 3 pseudo-Hurler polydystrophy cell lines) comprised one complementation group and 5 cell lines from patients with variant forms of pseudo-Hurler polydystrophy comprised a distinct complementation group. In the first group, total or partial deficiency of the transferase activity was demonstrated with both natural (lysosomal enzymes) and artificial (alpha-methylmannoside) acceptor substrates with low Vmax but apparently normal Km values for the donor (UDP-GlcNAc) and acceptor (alpha-methylmannoside) substrates. The activity toward artificial substrate could be inhibited by adding exogenous lysosomal enzyme preparations to the reaction mixture. In the second group, the cells demonstrated deficiency of the transferase activity toward lysosomal enzyme acceptors but had normal activity toward alpha-methylmannoside acceptor and this activity could not be inhibited by the addition of exogenous lysosomal enzyme preparations. These findings suggest that N-acetylglucosaminylphosphotransferase is composed of at least two distinct subunits, a catalytic subunit which is absent or defective in the first complementation group, and a recognition subunit which is altered or deficient in the second group.     

AAV载体介导的蓬佩病模型小鼠体内基因治疗研究*

Objective: Pompe disease is a lysosomal glycogen storage disease caused by acid α-glucosidase (GAA) deficiency, which is characterized by glycogen accumulation in the heart, skeletal muscle, and central nervous system (CNS). AAV vector-mediated gene therapy is expected to be a breakthrough in the treatment of Pompe disease. In this study, AAV9 vector was used to mediate GAA gene transfer in Pompe disease model mice, and the changes of GAA protease activity, glycogen accumulation in tissues and pathological changes in mice after transgenic intervention were evaluated. Methods: Codon optimized GAA gene (coGAA) was carried by AAV9 vector, and the AAV vector was packaged by baculovirus production process. Adult Pompe model mice were given a single intravenous injection at the dose of 1.1×10¹³, 3.0×10¹³, 1.2×10¹⁴ vg/kg, and aged Pompe model mice were given a single intravenous injection at the dose of 3.0×10¹³ vg/kg. After reaching the end point of the experiment, the mice were euthanized, GAA protease activity was determined by fluorescence spectrophotometry, glycogen accumulation was observed by PAS staining, and pathological changes were detected by HE staining. Results: Five weeks after administration, GAA protein was widely expressed in all tissues of adult model mice, with higher expression levels in heart and liver, and lower expression levels in brain and spinal cord. After rAAV9-coGAA treatment, glycogen content in myocardium, skeletal muscle and brain decreased, and vacuolar degeneration in myocardium and skeletal muscle decreased significantly. After treatment, the tissue enzyme activity of the aged animals was significantly increased compared with that of the model mice. The vacuolar degeneration and inflammatory cell infiltration of the myocardium were decreased, but the pathological improvement of skeletal muscle was limited. Conclusion: A single intravenous injection of rAAV9-coGAA can enhance GAA enzyme activity, reduce glycogen accumulation and improve pathology in Pompe model mice. The therapeutic effect was dose-dependent, and the injection also had certain therapeutic effect on aged animals. This study laid a theoretical foundation for the clinical application of AAV9 mediated gene therapy via intravenous route in Pompe disease.