8. Novel enzyme replacement therapy for Gaucher disease: Ongoing Phase III clinical trial with recombinant human glucocerebrosidase expressed in plant cells

Abstracts / Molecular Genetics and Metabolism 96 (2009) S12–S47 the potential to act as drugs when they can stabilize the native conformation of a mutant enzyme. In 2006 ExSAR applied for orphan drug designation from the FDA for pyrimethamine (PYR) for the treatment of late-onset GM2-Gangliosidosis. The application is pending the positive outcome of clinical trials. PYR is an FDA-approved drug which readily passes the blood–brain barrier (BBB). It is currently used to treat malaria and toxoplasmosis. Our invitro cell-based studies with fibroblasts from these patients show that PYR can function as a PC for Hex A and raise intracellular Hex A levels. Our hypothesis is that PYR administered to adult TSD and SD patients can improve neurological function and health. Here our objective is to assess the safety and tolerability of the drug. We will conduct a Phase I trial using PYR to treat patients affected with GM2-gangliosidosis. Hex A and B (12) activities in plasma and peripheral blood leukocytes will also be measured. Our working hypotheses are that PYR administered according to the regimen of the trial will be tolerated and safe and result in increased Hex A levels in blood and brain.

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we propose a two-hit’ model in which abnormal levels of GM1 at the ER membrane indirectly induces MMP via Ca2+-mediated signal released from the ER. Deciphering the precise mechanisms by which GM1 orchestrates neuronal cell death after it reaches a critical intracellular concentration will help to understand the pathophysiology of this disease and to design more efficacious therapies. (Supported in part by NIH Grants: GM60905, DK52025, Assisi Foundation of Memphis and ALSAC.) doi:10.1016/j.ymgme.2008.11.007

7. An efficient high-risk screening protocol for Fabry disease Christiane Auray-Blaisb, David S. Millingtona, Sarah P. Younga, Joe T.R. Clarkeb, Schiffmann Raphaelc, aDuke University Medical Center, Durham, NC, USA, bUniversiti de Sherbrooke, Sherbrooke, Quebec, Canada, cBaylor Research Institute, Dallas, TX, USA

doi:10.1016/j.ymgme.2008.11.005

5. Adeno-associated virus mediated gene therapy in a murine model of Morquio syndrome type A Carlos Almecigaa, Adriana Montaqob, Luis Bareraa, Shunji Tomatsub, aPontificia Universidad Javeriana, Bogota, Distrito Capital, Colombia, bSaint Louis University, Saint Louis, MO, USA Morquio A disease (MPS IVA) is an autosomal recessive disorder caused by the deficiency of N-acetyl-galactosamine-6-sulfate sulfatase (GALNS), leading to accumulation of keratan-sulfate and chondroitin-6-sulfate mainly in bone and cornea. As a first step towards the development of a gene therapy for MPS IVA, we used adenoassociated virus derived-vectors for the evaluation of two variables: (a) the effect of the promoter region on GALNS expression, and (b) the effect of the co-expression with the Sulfatase Modifying Factor 1 (SUMF1) gene on the enzyme activity. The results showed that in HEK293 cells the eukaryotic promoters human alpha-antitrypsine (AAT) and human elongation factor 1-alpha (EF1) allowed similar GALNS activity levels than those in cells transfected with a vector containing the cytomegalovirus (CMV) promoter. GALNS activity in the lysated from cells cotransfected with SUMF1 was increased up to fourfold and allowed the enzyme activity detection in the culture media. In-vivo the enzyme activity in plasma and tissues from MPS IVA adult mice infused with AAT-GALNS was around 40% of wild-type levels after 12 weeks postinfusion. These GALNS levels were increased around twofold in animals coinfused with GALNS and SUMF1. GALNS activity in bone from animals infused with AAT-GALNS was not observed while coinfusion with the SUMF1 vector allowed a significant increment in the enzyme activity. No difference in urine GAG levels was observed between MPS IVA untreated, MPS treated and wild-type animals, showing the need to use a different biomarker or quantification method. In summary these results are the first evidence of the enzymatic correction in a murine model of MPS IVA by gene therapy and show the advantage of the co-expression with SUMF1 in order to obtain therapeutic levels of enzyme activity.

Introduction: Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results in fatty deposits in cells of various tissues and body fluids. Urinary accumulation of globotriaosylceramide (Gb3) in Fabry patients is a reliable diagnostic biomarker. Misdiagnosis of Fabry disease is frequent: it takes on average 13.7 years from the onset of symptoms to diagnosis confirmation in hemizygotes and 16.3 years in heterozygotes. We hypothesize that alpha-galactosidase A deficiency is a modifiable general cardiovascular risk factor and that patients with unrecognized Fabry disease presenting with renal, cardiac or cerebrovascular complications are identifiable by a simple, screening process in the population at large. We present a protocol to screen for Fabry disease in at-risk populations attending cardiology, stroke and nephrology clinics. Method: Patients will be evaluated for the presence of specific clinical criteria related to renal and cardiac abnormalities. All patients with vascular complications irrespective of age or other etiologic factors should be included. Screening will be performed using a high-throughput tandem mass spectrometric-based multiplex assay for urinary filter paper Gb3/creatinine. This methodology detects both affected males and females reliably at low cost, except those with variants with increased residual enzyme activities associated with near-normal Gb3 excretion. Results: Abnormal Gb3 excretion in males would trigger alpha-gal A enzyme assay in plasma, peripheral blood leucocytes or dried blood spots. Hemizygotes with marked deficiency of enzyme activity would undergo GLA mutation analysis to confirm the diagnosis of Fabry disease, while women with abnormal Gb3 excretion would be subjected to GLA mutation analysis only. Conclusions: The specific aim of this protocol is to detect Fabry patients rapidly after their first referral to specialized clinics or hospitals to allow early therapy. This protocol is potentially applicable worldwide. doi:10.1016/j.ymgme.2008.11.008

8. Novel enzyme replacement therapy for Gaucher disease: Ongoing Phase III clinical trial with recombinant human glucocerebrosidase expressed in plant cells

doi:10.1016/j.ymgme.2008.11.006

6. Accumulation of GM1-ganglioside in ER-mitochondrion microdomains elicits mitochondrial Ca2+ overload and neuronal apoptosis Ida Annunziata, Renata Sano, Annette Patterson, Alessandra d’Azzo, St. Jude Children’s Research Hospital, Memphis, TN, United States Gangliosides are emerging as important determinants of apoptosis in physiological and pathological conditions. In the lysosomal storage disease GM1-gangliosidosis, progressive accumulation of GM1-ganglioside (GM1), due to deficiency of acid beta-galactosidase (beta-gal), is associated with neurodegeneration. We have previously demonstrated that in the mouse model of this disease, abnormal buildup of GM1 at the ER membrane induces depletion of ER Ca2+ stores and activation of the unfolded protein response (UPR), resulting in neuronal apoptosis (Tessitore etal., 2004). Given the cross-talk between ER and mitochondria, we now asked whether GM1-mediated disturbance of Ca2+ homeostasis could directly or indirectly influence mitochondrial function, and involve this organelle in the apoptotic process. We found that GM1 progressively accumulates in mitochondria-associated membranes (MAMs) and likely influences Ca2+ exchange between the ER and the mitochondria at these sites. A GM1-dependent mitochondrial Ca2+ overload induces mitochondrial membrane permeability (MMP), dissipation of the membrane potential, opening of the permeability transition pore and release of apoptogenic factors, which ultimately activate an apoptotic caspase-cascade (d’Azzo etal., 2006). Based on our data,

David Aviezera, Einat Almon-Brilla, Yoseph Shaaltiela, Gad Galilid, Raul Chertkoffa, Sharon Hashmuelia, Eithan Galunc, Ari Zimranb, aProtalix Biotherapeutics, Carmiel, Israel, bGaucher Clinic, Shaare Zedek Medical Center, Jerusalem, Israel, cGoldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel, dPlant Sciences, Weizmann Institute of Science, Rechovot, Israel Gaucher disease, characterized by glucocerebrosidase (hGCD) deficiency, provokes glucosylceramide accumulation in cellular lysosomes. Disease clinical pathology includes anemia, thrombocytopenia splenomegaly, skeletal pathology and pulmonary hypertension/infiltration. Current therapy uses mammalian based production of recombinant glucocerebrosidase for enzyme replacement therapy (ERT) that involves post-expression glycan remodeling for exposing mannose structures, required for uptake by Macrophages. Protalix has developed a propriety plant cell expressed active form of rh-glucocerebrosidase (prGCD). Protalix’s unique technology permits control of the glycosylation pattern and consistency through targeting expression to specific plant cell organelles. Hence, prGCD has intrinsic exposed mannose residues and demonstrates batch to batch consistency. prGCD exhibits similar crystal structure and biological activity to that of the currently used CHO expressed imiglucerase (Cerezyme) using in-vitro assays. Preclinical toxicology studies showed no treatment-related adverse events, no neutralizing antibodies and no clinical findings. Phase I safety clinical trial showed that prGCD administered intravenously in sequential doses (15, 30 and 60 U/kg) was well tolerated, all tests being within normal ranges, with no treatment related adverse events. Pharmacokinetic analysis demonstrated a prolonged half life. All immunological specific tests were within normal ranges. An international multi-center Phase III Pivotal trial is currently ongoing under FDA Special Protocol Assessment

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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