13. Enzyme replacement therapy with Alglucosidase alfa in juvenile-adult glycogenosis type 2 patients

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Abstracts / Molecular Genetics and Metabolism 96 (2009) S12–S47

approval where 30 untreated patients will be administered with 30 U/kg or 60 U/ kg per infusion over a period of 9 months. Following completion of the protocol, patients are offered to enter an on-going Extension study. In addition, a switch-over study to prGCD is to begin soon. doi:10.1016/j.ymgme.2008.11.009

9. Potential combination therapies stemming from strategies against LSDs and PADs Ben A. Bahr, David Butler, Jeannie Hwang, Candice Estick, University of Connecticut, Storrs, CT, United States Accumulation of un-degraded substrates lead to CNS effects in lysosomal storage disorders (LSDs). Intracellular accumulations also occur in Alzheimer’s and other protein accumulation disorders (PADs). Strategies exist or are in development for treating age-related disorders, and the therapeutic potential of LSD treatments may be enhanced by such strategies, including: (1) anti-inflammatory drugs thought to reduce progression of pathology, (2) glutamatergic regulatory drugs thought to reduce excitotoxic contribution, (3) positive modulators of synaptic signaling to promote neuronal activity, (4) transport/microtubule stabilizers to support microtubule functionality, or (5) cellular clearance enhancers. The latter stems from the fact that altered lysosomes and autophagic trafficking occur in LSDs, PADs, and normal aging. Activators of macroautophagy have been shown to promote clearance of toxic protein assemblies. Alternatively, we found that lysosomal modulators (e.g., Z-Phe-Ala-diazomethylketone PADK) markedly increase activated cathepsins levels 2- to 9-fold invitro and in AD mouse models. Reducing protein accumulation is essential for slowing AD progression, thus we showed that PADK treatment that produced a dosedependent increase in cathepsins B and D in the hAPP mice also caused clearance of intracellular Abeta and early plaque formation. PADK also increased synaptic markers GluR1, synapsin II, and synaptophysin to control levels in hippocampus and other brain regions. Correspondingly, both synaptic and behavioral deficits were attenuated, for instance the treated mice exhibited improved performance in a Y-maze test for the hippocampal-dependent task of spontaneous alternations. These results indicate that promoting lysosomal processes can ameliorate Abeta-induced disruption of cellular and functional integrity, and may represent an effective strategy for treating AD and other disorders involving pathogenic accumulations. Assessment of common pathogenic processes involved in cellular accumulation stress may warrant investigations into beneficial synergistic effects mediated by novel combination therapies for LSDs. doi:10.1016/j.ymgme.2008.11.010

10. Impairment of autophagy in lysosomal storage diseases Andrea Ballabio, Fondazione Telethon, Italy, Federico II University, Naples, Italy Autophagy is the mechanism responsible for the turn-over of intracellular organelles and digestion of protein aggregates which are sequestered by autophagosomes and degraded upon the fusion of the autophagosome with the lysosome. Several neurodegenerative disorders, such as Alzheimer, Parkinson and Huntington diseases are associated with an impairment of autophagy. We have analyzed the autophagic pathway in two different murine models of lysosomal storage disorders (LSD), Multiple sulfatase deficiency (MSD) and mucopolysaccharidosis type IIIA (MPS IIIA). Western blotting, immunofluorescence and immunohistochemical analyses using anti-LC3 antibodies demonstrated a significant intracellular accumulation of autophagic (LC3-positive) vacuoles in MEF as well as in several brain regions of both MSD and MPS IIIA mice. Accumulation of autophagosomes was also confirmed by ultrastructural analysis. Co-staining of MEF using both anti-LC3 and anti-LAMP2 antibodies demonstrated that autophagosomes do not co-localize with lysosomes, suggesting the presence of a fusion defect. As a consequence of an impairment of autophagy, a massive intracellular accumulation of ubiquitin-positive aggregates and an increased number of mitochondria with altered membrane potential were detected in the brain of both MSD and MPS IIIA mice. Interestingly, the build-up of polyubiquitinated proteins and dysfunctional mitochondria has been associated with neuronal cell death in neurodegenerative diseases. Taken together our data indicate that accumulation of storage material, due to the lysosomal enzyme deficiency, causes a lysosomal dysfunction which affects the autophagic pathway, and more specifically the formation of autophagolysosomes. We postulate that neurodegeneration in LSD is caused by secondary storage of toxic protein aggregates due to an impairment of autophagy. doi:10.1016/j.ymgme.2008.11.011

11. Increasing expression of glucosylceramide-synthesizing enzyme in mouse models of Gaucher disease Sonya Barnes, You-Hai Xu, Gregory Grabowski, Cincinnati Children’s Hospital Research Foundation, University of Cincinnati College of Medicine, Cincinnati, OH, United States Gaucher disease (GD) is a lysosomal storage disease caused by deficient acid betaglucosidase [GCase] enzyme activity. This deficiency leads to accumulation of toxic substrates mainly glucosylceramide (GC) in cells of monocytic origin, mainly macrophages. Several viable models of GD have been generated by introducing point mutations into the mouse GCase (gba) locus, including N370S, V394L, D409H, or D409V. These mouse models share only some characteristics of the human phenotypes. We hypothesized that increasing GC levels in such mice might produce phenotypes more closely resembling those in humans. Thus, we created transgenic mice overexpressing the glucosylceramide-synthesizing enzyme, UDP-glucose: ceramide glucosyltransferase (GCS), that were then bred into a D409V/null background. RT-PCR and Northern analyses showed high level expression of the GCS transgene (tg) mRNA in brain, liver, lung, and spleen. Since the GCS knockout (GCS/) mice is an embryonic lethal, we rescued this lethal phenotype through expression of the GCS transgene (tg/GCS/) under the control of the ubiquitous ROSA promoter; thus demonstrating a functional transgene invivo. D409V/null mice overexpressing GCS (tg/GCS+/+) had a neonatal lethal phenotype with death within 24 h. Lipid analyses of these mice show GC levels comparable to those in GCase null mice. The use of several other GCase mutant mice with this GCS tg should be useful in understanding the complexity of Gaucher disease and for determining the threshold level of substrate flux that influences phenotypic development. doi:10.1016/j.ymgme.2008.11.012

12. Suppression of a nonsense mutation in a mouse model of Hurler syndrome David Bedwella, Dan Wanga, Charu Shuklaa, Xiaoli Liua, Shirley Yehb, Ellen Welchb, Trenton Schoeba, Kim Keelinga, aUniversity of Alabama at Birmingham, Birmingham, AL, United States, bPTC Therapeutics, Inc., South Plainville, NJ, United States The mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by a deficiency of various enzymes catalyzing the degradation of glycosaminoglycans (GAG). MPS I is characterized by the deficiency of alpha-L-iduronidase (encoded by the IDUA gene) and the accumulation of dermatan and heparan sulfate. Hurler syndrome, the most severe form of MPS I, is a progressive disorder with multiple organ involvement and patients usually die within first decade if left untreated. Two nonsense mutations of the IDUA gene, W402X and Q70X, account for 70% of MPS I mutations in the Caucasian population. Recently, compounds such as aminoglycosides and PTC124 have been shown to facilitate the incorporation of near-cognate tRNAs at the nonsense mutation during translation and suppress premature translation termination that causes human genetic diseases such as Duchenne muscular dystrophy and cystic fibrosis. Consistent with these results, we have shown that the aminoglycoside gentamicin suppresses nonsense mutations in the IDUA mRNA and restores enough alpha-L-iduronidase activity to eliminate abnormal GAG accumulation in fibroblasts from a patient affected with Hurler syndrome. We developed a targeted knock-in mouse model carrying the nonsense mutation corresponding to the human IDUA-W402X mutation to further evaluate this novel treatment strategy. The initial characterization showed that this Idua-W402X mouse has biochemical and morphological defects consistent with those found in Hurler syndrome patients. The alpha-L-iduronidase activity was almost undetectable in all organs examined. The urinary and tissue GAG were significantly elevated. Finally, histological examination and electron microscopy revealed vacuole-like accumulation characteristic of the lesions seen in Hurler syndrome patients. After treatment with drugs that induce readthrough, homozygous IDUA-W402X mice showed increased enzyme activity and significantly reduced GAG accumulation, suggesting that nonsense codon suppression could be a novel treatment strategy for patients with Hurler syndrome. doi:10.1016/j.ymgme.2008.11.013

13. Enzyme replacement therapy with Alglucosidase alfa in juvenile-adult glycogenosis type 2 patients Bruno Bembia, Sabrina Ravagliab, Federica Edith Pisaa, Giovanni Cianac, Agata Fiumarad, Marco Confalonierie, Rossella Parinif, Miriam Rigoldif, Arrigo Mogliab, Alfredo Costab, Cesare Danesinog, Maria Gabriela Pittisc, Andrea Dardisa,c, aRegional Coordinator Centre for Rare Diseases, University Hospital Santa Maria della Misericordia, Udine, Italy, bInstitute of Neurology, I.R.C.C.S. C Mondino of Pavia, Italy, cMetabolic Unit I.R.C.C.S. Burlo Garofolo of Trieste, Italy, dPaediatric Clinic, University of Catania, Italy, e Pulmonary Unit, University Hospital of Trieste, Italy, fMetabolic Unit, Paediatric Dept., San Gerardo Hospital, Monza, Italy, gMedical Genetics, University of Pavia, Italy

Abstracts / Molecular Genetics and Metabolism 96 (2009) S12–S47 Glycogenosis 2 (G2), a lysosomal storage disorder due to acid alfa-glucosidase (GAA) deficiency, has recently been demonstrated to be responsive to human recombinant (rhGAA) enzyme replacement therapy (ERT) in infantile phenotypes. To date, no large studies have been published on juvenile-adult phenotypes. An independent multi-centric Italian study, supported by the Agenzia Italiana del Farmaco (AIFA), was proposed in 2006 to verify ERT effectiveness in late-onset phenotypes. Twenty-nine patients (13 females, 16 males; 7 juveniles, 22 adults) were enrolled; they received a bi-weekly rhGAA infusion of 20 mg/kg, and were monitored for: general conditions, respiratory function, ventilatory support, 6-min walking test (6MWT), Walton scale (WS), muscular imaging. Biochemistry included: CK, LDH, AST, ALT, blood pCO2. After 12 months of ERT, tracheotomy was removed in 3 of the 4 carrying patients (no further ventilatory support was needed in 2); patients’ mean ventilation time decreased from 15.5 to 9.6 h. All patients improved in their 6MWT (p < 0.0001) and WS severity decreased in 5 patients (p = 0.0039). Headache and muscle pain affecting, respectively, 27.6% and 37.9% of patients at baseline persisted in 6.9% and 10.3% of them after one year. A statistically significant reduction of CK (p = 0.0075), LDH (p = 0.0003), AST (p = 0.0028), ALT (p = 0.0023) was observed. No patient showed severe secondary effects to ERT. Conclusions: Human recombinant GAA therapy demonstrated to be safe and capable to improve clinical symptoms and laboratory parameters in treated patients. Results showed to be independent of patient age and disease severity.

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ades, specific functions of these proteins have only recently begun to be recognized. Although some of the normal functions of LAMP-1 can be substituted by the structurally related LAMP-2, LAMP-2 itself has more specific tasks. The specific deletion of the LAMP2 gene in mice (LAMP2-knockout mice) has revealed roles for LAMP-2 in lysosomal enzyme targeting, macroautophagy and lysosomal biogenesis. LAMP-2 also excerts a specific role in lysosomal cholesterol export. Extensive accumulation of autophagic vacuoles in LAMP-2 deficient tissues indicate a role of LAMP-2 in biogenesis of these vesicles. Additionally, LAMP-2A is reported to be the receptor for chaperon mediated autophagy (CMA), a selective mechanism for the degradation of cytosolic proteins within lysosomes. Many neurodegenerative diseases like lysosomal storage disorders, Parkinson disease (PD) and Alzheimer disease underline the importance of CMA and macroautophagy (MA) for normal brain function. Previous analyses have shown that both autophagic pathways, CMA and MA contribute to alpha-synuclein degradation which is thought to be central in the pathogenesis of Parkinson’s disease. Deficiency of LAMP-2 in humans leads to Danon disease, a fatal cardiomyopathy and myopathy associated with mental retardation. However, the neuropathology of these patients has not yet been investigated. To get insights into the neuronal functions of LAMP-2 we have studied the consequences of LAMP-2 deficiency in brains of LAMP2 Knockout mice by histological and biochemical methods. Preliminary results show altered expression of autophagic and lysosomal marcer proteins as well as changes in lipid composition. With this presentation we will provide first insights into the neuronal pathology of LAMP-2 Knockout mice which has not been reported so far.

doi:10.1016/j.ymgme.2008.11.014 doi:10.1016/j.ymgme.2008.11.016

14. A natural history study of hexosaminidase deficiency Kendra J. Bjoraker, Chester B. Whitley, Charnas Lawrence, University of Minnesota, Minneapolis, MN, United States Tay-Sachs disease, Sandhoff disease and late-onset Tay-Sachs disease (LOTS) are fatal genetic disorders in which harmful quantities of a fatty substance called ganglioside GM2 accumulate due to insufficient activity of an enzyme hexosaminidase. Currently, there is no treatment. Three sub-studies are proposed. Study 1 will focus on the developmental course of the spectrum of infantile Tay-Sachs disease from retrospective data. Study 2 will gather longitudinal prospective data from patients with infantile disease seen at the University of Minnesota to delineate the natural history of Tay-Sachs. Study 3 will be a longitudinal study of LOTS to understand the progression of CNS disease using quantitative methods of neuroimaging and neuropsychological measures. Objectives: Aim 1: Develop an index of disease progression in infantile Tay-Sachs disease through: (A) first, a retrospective medical chart/record review for clinical data points. Second, participant caretakers of infants with Tay-Sachs disease will be asked to complete the University of Minnesota Natural History Study Questionnaire for Infantile Tay-Sachs Disease (MNQSTD). (B) Prospective: We will collect longitudinal neuropsychological and neuroimaging data in patients with infantile Tay-Sachs disease to characterize the developmental course and longitudinally examine individual growth trajectories. Predictors will include mutation type, age at diagnosis, biomarkers and surrogate markers such as MRI results. Aim 2: To better understand the underlying CNS structure and function abnormalities in LOTS disease and to measure change in function over time through neuroimaging and neuropsychological data. Methods: Infantile: Recruit 5 new patients the 1st year, 10 the 2nd year, and 10 the 3rd year. Each child will be seen yearly for a total of at least 3 or possibly 4 follow-up visits within the 5 year period. LOTS: Participants: Recruit 5 new patients the 1st year and 10 new patients the 2nd year. Each patient will be seen once a year. Both Infantile and LOTS: quantitative neuroimaging, biomarkers, and neuropsychological protocol will be used to collect data as well as relevant medical records. Currently, there is no treatment for Tay-Sachs disease, and no studies have documented the longitudinal natural history of either the early or late onset disease. The data will provide end-points for future therapies, so as to guide medical decisions about treatment and provide objective measurement of treatment outcomes. This information will help objectify disease progression to create a disease stage and severity scale to assist in diagnosis and evaluation of the outcomes of medical treatment of these children. The overall long term goal of this research is to identify brain and behavior abnormalities that can both predict course and be sensitive to change resulting from treatment. doi:10.1016/j.ymgme.2008.11.015

15. Role of LAMP2 in neuronal autophagy Judith Blanza, Steven U. Walkleyb, Paul Saftiga, aUniversity of Kiel (Germany), kiel, Germany, bAlbert Einstein College of Medicine (NY, USA) LAMP-2 and LAMP-1 are abundant proteins of the membranes limiting late endosomes and lysosomes. Even though the existence of these proteins is known for dec-

16. Cardiac risk stratification for individuals with MPS IH-S: Where we are today Elizabeth Braunlin, Chester Whitley, University of Minnesota, Minneapolis, MN, United States The clinical findings in MPS I syndrome form a continuum from presentation in infancy and death within the first decade of life (MPS IH) to discovery in adulthood and survival well into middle age (MPS IS). Individuals with MPS IH-S are thought to have clinical findings lying somewhere in the middle of this continuum. The natural history of MPS IH-S is not well studied; nor are the cardiac findings from autopsy well described. Individuals who receive enzyme replacement therapy (ERT) come from precisely this subset of patients. Sudden, unexpected death has been reported in untreated MPS, and may be related to coronary artery occlusion. Individuals with MPS IH-S have died unexpectedly after months or years of ERT. Although coronary artery pathology in untreated MPS IH is well described, the coronary pathology of MPS IH-S has been rarely described and even then, without pictures. Individuals with the MPS syndromes often undergo a cardiac evaluation prior to invasive procedures. This usually includes clinical evaluation, resting ECG and cardiac ultrasound. A normal resting ECG and ECHO will not exclude the presence of substantial coronary artery pathology. A better method of stratifying coronary artery risk is needed. Noninvasive exercise imaging testing is a low-risk procedure commonly used to detect suspected atherosclerotic coronary artery disease in adults. A recent large meta-analysis in this population concludes that a normal exercise imaging test is an excellent negative predictor for subsequent cardiac events. The prospective performance of exercise or dobutamine ultrasound imaging before initiation of ERT in individuals with MPS IH-S, may provide the least invasive and most reliable method at present to identify those at risk for subsequent cardiac events. doi:10.1016/j.ymgme.2008.11.017

17. Biological variation and sample collection effects on alpha-galactosidase A enzyme measurements in Fabry and non-Fabry populations Marsha Browninga, Petra Olivovae, Kristen van der Veenb,d, X. Kate Zhange, Emmaline Cullene, Michael Rosee, Katherine Simsb,c,d, Joan Keutzere, aBoston, MA, United States, b Lysosomal Disorders Unit, Massachusetts General Hospital, Boston, MA,, cHarvard Medical School, dMGH Center for Human Genetic Research, Boston, MA,, eGenzyme, Framingham, MA Background: Fabry disease is an X-linked lysosomal storage disorder due to an enzyme deficiency of alpha galactosidase A (AGAL, EC 3.2.1.22) resulting in multi-systemic organ dysfunction. Despite increasing utilization of dried blood spot enzyme assay measurements for AGAL, there is not a published experience concerning the effects of blood sample collection techniques on final enzyme activity in a fluorometricbased AGAL assay. Methods: DBS samples were prepared either by spotting blood collected into an ethylenediaminetetraacetic acid (EDTA) tube or by direct spotting of blood from a fin-