The evaluation of a loss-of-function GBA variant found in Gaucher patients with Parkinson disease

Abstracts / Molecular Genetics and Metabolism 102 (2011) S3–S47

AAV9 to cross the BBB from the vasculature to achieve long-term global CNS, and widespread somatic restoration of α-N-acetylglucosaminidase (NAGLU) activity. A single intravenous (IV) injection of rAAV9-CMVhNAGLU, without extraneous treatment to disrupt the BBB, restored NAGLU activity to normal or above normal levels in adult MPS IIIB mice, leading to complete, or near complete, correction of lysosomal storage pathology in the CNS and periphery, and correction of astrocytosis and neurodegeneration. The IV-delivered rAAV9 vector also transduced abundant neurons in the myenteric and submucosal plexus, suggesting peripheral nervous system targeting. While CNS entry did not depend on osmotic disruption of the BBB, it was significantly enhanced by pretreatment with an IV infusion of mannitol. Most important, we demonstrate that a single systemic rAAV9-NAGLU gene delivery provides long-term (>18 month) neurological benefits in MPS IIIB mice, resulting in significant improvement in cognitive and motor function, and extension of survival. These data suggest promising clinical potential using the trans-BBB neurotropic rAAV9 vector for treating MPS IIIB and other neurogenetic diseases. We anticipate that this minimal invasive procedure will pose a minimal burden to MPS IIIB patients and is well suited for clinical application. doi:10.1016/j.ymgme.2010.11.063

Gaucher disease as a macrophage activation disorder Ozlem Goker-Alpan, Praveena Karamthoti, Oral Alpan, Center for Clinical Trials, Springfield, VA, USA Although macrophages protect against opportunistic infections, when they become inappropriately activated, they can participate in the development of autoimmune and inflammatory diseases. Macrophage activation depends on the products of T helper and natural killer (NK) cells. In Gaucher Disease(GD), there is not only a deficiency of cellular and humoral immune functions, but also the dysfunction of monocyte-macrophage system. Gaucher cells strongly express markers of alternative activation such as CCL18 and chitotriosidase. We hypothesized that in GD, the mechanisms leading macrophage activation is caused by not only lipid accumulation, but also through the effects of immune system elements. In a cohort of 15 GD patients, 20% had clinical evidence of macrophage activation, in whom T and B cell subsets, NK cell numbers and activity were evaluated in peripheral blood mononuclear cells. NK cell function was measured by cadmium lysis assay. NK, T and B cell subsets were evaluated by flow cytometry. Chitotrisidase levels were followed as a marker of alternative macrophage activation. While, all displayed nonspecific T cell abnormalities, and increased B cell numbers, a subgroup of patients had decreased number of NK cells or a significantly impaired in vitro target killing. While, ERT led to a decrease in chitotriosidase, inflammatory markers in peripheral blood remained elevated. Innate immune dysfunction could be additive to glucosylceramide accumulation to induce macrophage activation. In GD patients who exhibit a suboptimal response to therapy, macrophage dysregulation and NK cell dysfunction should be explored. doi:10.1016/j.ymgme.2010.11.064

Enzyme replacement therapy corrects Lysosomal Acid Lipase Deficiency (LALD) in a mouse model of Wolman disease/cholesteryl ester storage disease Gregory Grabowskia, Hong Dua, Brian Pescatoreb, Alla Romashkob, Muthu Meiyappanb, Paolo Martinib, Lawrence Charnasb, Alicia Gomez-Yafalb, You-Hai Xua, Oluwaseyi Westmorelanda, Lori Stantona,

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Gregory Grabowskia, aCincinnati Children's Hospital Medical Center, Cincinnati, OH, USA, bShire Human Genetic Therapies, Boston, MA, USA Lysosomal acid lipase (LAL) hydrolyzes triglycerides (TGs) and cholesteryl esters (CEs); Lysosomal Acid Lipase Deficiency (LALD) causes either an infantile form known as Wolman disease (iLALD) or a later onset form, known as cholesteryl ester storage disease (CESD or loLALD). The LAL KO mouse model (lal-/-) resembles human LALD with storage of CEs and TGs in multiple organs, and loss of subcutaneous and omental fat. Recombinant human LAL (rhLAL) was expressed in the human cell HT1080 and purified from culture media. Purified rhLAL has a molecular weight ~54.5 kDa, decreasing to ~ 43 kDa after deglycosylation by PNGase F. Three rhLAL doses - 24U, 48U and 96U (3.2, 6.4, and 12.8 mg/kg) were tested in lal-/- mice for pharmacokinetics by i.v. administration. The t1/2 values in sera were: 10 min for 3.2 and 6.4 mg/kg and 15 min for 12.8 mg/kg (n = 5). The 6.4 mg/kg dose demonstrated a t1/2 of 5 hr in the liver. The t1/2 of both rhLAL enzyme activity and rhLAL protein in serum and in tissues are comparable, suggesting the absence of inactivated protein in both serum and tissues. Efficacy of two different doses (0.8 mg/kg and 3.2 mg/kg) were administrated though tail vein bolus injection weekly for ten weeks in both young (2 mos, n = 25) and old (4 mos, n = 17) lal-/- mice. Compared to age matched lal-/- control mice (n = 14), i.v. rhLAL reduced liver weight (20-26% with 0.8 mg/kg dose, 39-42.0 % for 3.2 mg/kg) and spleen weight (31-42% for 0.8 mg/kg and 36–46.2% for 3.2 mg/kg) in both 2 and 4 mos old lal-/- mice. Histologic analysis of liver, spleen, small intestine, adrenal gland and lymph node showed significant improvement with both doses, with reversal of phenotype at the highest dose in the 2 mos old mice. Tissue and plasma lipid levels, e.g. Cholesteryl esters, triglycerides and free cholesterol improved. These studies demonstrate proof of concept for ERT with rhLAL for human LAL deficiency.

doi:10.1016/j.ymgme.2010.11.065

The evaluation of a loss-of-function GBA variant found in Gaucher patients with Parkinson disease Ann Marie Gustafson, Arash Velayati, Yotam Blech-Hermoni, Jae Choi, Wendy Westbroek, Claudia Landazabal, Ehud Goldin, Barbara Stubblefield, Ellen Sidransky, Nahid Tayebi, NIH, NHGRI, Bethesda, MD, USA Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene (GBA). Deficiency of the enzyme glucocerebrosidase (GCase) leads to accumulation of glucosylceramide. Recent studies have shown that Parkinson disease (PD) patients are over five times more likely to carry GBA mutations than controls. The Ashkenazi Jewish GBA founder mutation c.84dupG, was detected with a frequency of 2.1% among Ashkenazi PD patients compared to 0.27% in controls. The G insertion results in a downstream stop codon generating a putative 25 amino acid peptide. Homozygous c.84dupG mutations are presumed to be lethal. In this study, we evaluated the expression and toxicity of the putative peptide as well as the brain pathology of two c.84dupG carriers with PD. First, we excluded the occurrence of a potential alternative exonic splice site by RT-PCR and sequencing of GBA alleles of (c.84dupG; N370S) patients. An in vitro translation assay showed no putative peptide translation. Confocal microscopy and western blotting on (c.84dupG;N370S) cells and protein extracts with an custom-made antibody directed against the c.84dupG peptide showed no endogenous expression of the peptide. No morphological changes or

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Abstracts / Molecular Genetics and Metabolism 102 (2011) S3–S47

increased cell death were observed in CHO cells transfected with a c.84dupG construct. Interestingly, studies on brain samples from carriers with PD showed accumulation of GCase in Lewy bodies. In conclusion, our studies show that c.84dupG is a null mutation, suggesting that reduction in the total amount of GCase or enzymatic activity could contribute to the development of parkinsonism in carriers of this mutation.

doi:10.1016/j.ymgme.2010.11.066

CD77 as a biomarker for effectiveness of enzymatic replacement therapy in Fabry disease

including decreasing hepatomegaly and continued linear growth at the 75th percentile. MPS quant values decreased initially but plateaued in the range 200-300 mg MPS/g creatinine. His antibody response has been no IgE response, moderate IgG response and a large neutralizing antibody response. The challenge becomes addressing the effectiveness of 0.5 mg/kg/week in the face large amounts of neutralizing antibodies, and the most clinically relevant biomarkers to use in monitoring efficacy of ERT. A question to be addressed over time is will the pearls and other systemic GAG deposits resolve in the face of large neutralizing antibodies. Functional ability, quality of life and trajectory of the natural history of MPS II in light of early ERT will be outcome measures important in the clinical care of patients with MPS II receiving ERT. doi:10.1016/j.ymgme.2010.11.068

Semiramis Hadad, Ester Miranda Pereira, Adalberto Socorro da Silva, Anaregina de Sousa Araújo, Henrique César Saraiva de Arêa Leão Costa Filho, Fernando Ferraz do Nascimento, Raimundo Nonato da Silva, Universidade Federal do Piauí, Teresina, Piauí, Brasil Fabry Disease (FD) is a disorder caused by mutations in the gene encoding for lysosomal enzyme -galactosidase A (-GAL). Reduced αGAL activity leads to progressive accumulation of glycosphingolipids, mostly, globotriaosylceramida (Gb3), within lysosomes. The genetic basis for disease development is extremely heterogeneous, and there are currently 473 mutations described for this condition. The currently accepted treatment for DF is the -GAL Enzyme Replacement Therapy (ERT), administered intravenously in a manner dependent on weight. One of the biggest challenges nowadays is to find a way to monitor the therapy effectiveness. The recent report of increased expression of CD77 in blood cells of patients with FD creates opportunity for use of this molecule as a potential marker for monitoring ERT. The goal of this study was to evaluate the expression of CD77 on the surface of the cells from Patientes carrier of FD (V269M mutation). For this, we compared the expression of CD77 in blood cells from 19 patients (from the same family, 6 men and 12 women) and 30 health controls, using flow citometry. We have conduced this studied for two months and our preliminar results points to a significant difference (p = 0,001) in CD77 expression between FD patients ( MFI = 814) and controls ( MFI = 191). Moreover, we found the levels of CD77 in FD patients under ERT are constantly decreasing. From our preliminar results we conclude that CD77 molecule is a promising tool assisting researchers in ERT monitoring in the context of Fabry Disease.

Oxidative stress as a therapeutic target in GLD Jacqueline Hawkins, Adarsh S. Reddy, Mark S. Sands, Washington University, St. Louis, MO, USA Globoid Cell Leukodystrophy (GLD) is a lysosomal storage disorder, resulting from the genetic deficiency of Galactosylceramidase (GALC). Loss of GALC causes accumulation of a toxic metabolite, Psychosine (Psy), which in turn causes oligodendrocyte death and demyelination. Elevation of oxidative stress markers has been demonstrated in oligodendrocytes exposed to Psy. Oligodendrocytes also may be spared from Psy-induced death when concurrently exposed to the antioxidant N-acetyl cysteine (NAC). We investigated oxidative stress markers in the nervous system of the Twitcher Mouse, the mouse model of GLD, and found them elevated compared to normal controls. Oxidative stress can lead to a myriad of problems within a cell, including protein, nucleic acid and lipid damage, and cell death. We sought to treat this stress by administering NAC to Twitcher mice, alone and in combination with bone marrow transplant, the standard treatment for GLD. We hypothesized that reducing the oxidative burden in the nervous system would protect against Psy toxicity, thereby improving oligodendrocyte survival and the clinical course of disease. Twitcher mice were treated with NAC continuously in the water, and also received a bolus of NAC three times per week IP. A cohort of animals also received BMT on postnatal day 2-4. Neither treatment resulted in an improvement in lifespan or behavioral markers of disease. While oxidative stress has been repeatedly demonstrated both in vitro and in vivo, it is likely that it is a secondary effect of disease, not a causative therapeutic target.

doi:10.1016/j.ymgme.2010.11.067 doi:10.1016/j.ymgme.2010.11.069 Pearls: Unusual presentation and clinical information in a young MPS II Patient Susan Hale, Seattle Children's Hospital, Seattle, WA, USA Mucopolysaccharidosis, Type II,( MPS II) is an X-linked recessive lysosomal disorder in which two glucosaminoglycans (GAG), dermatan sulphate and heparin sulphate, which accumulate in the tissues throughout the body due to deficiency of iduronatate sulphatase. Over the first year of 0.5 mg/kg/week idursulfase, a 3-year-old African American MPS II patient developed many cutaneous accumulations of GAG bilaterally across his shoulder blades and upper arms. This was an unexpected and perplexing development. Family questioned these growing pearls in light of enzyme replacement therapy (ERT) and efficacy of a year's worth of care. Anecdotal experience reported decrease in accumulation in patients who had later onset of ERT. Other clinical features of MPS II showed improvement in this patient,

Urine glycosaminoglycan: Exploring its role as an outcome measure for evaluating treatment response in the mucopolysaccharidoses Jonica Hazaert, Kyle Rudser, Chester Whitley, Jeanine Utz, University of Minnesota, Minneapolis, MN, USA Background: Urine glycosaminoglycan (GAG) has long been established as the diagnostic screening biomarker for the mucopolysaccharidoses (MPS). The role of GAG in monitoring patient response to MPS therapies, modifying MPS therapies, and comparing response between different therapies, such as hematopoietic stem cell transplant (HSCT) and intravenous enzyme replacement therapy (ERT), has not been defined. Reasons for this include variation in the following: 1) laboratory methods for processing GAG samples; 2) units of measure used to express GAG; 3) age-related GAG ref-