20. Adipose stem cell-mediated therapy for Krabbe disease

S16

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

ger prick or a venipuncture syringe. The activity of lysosomal alpha-galactosidase A was measured quantitatively by detecting the fluorescence of 4-methylumbelliferone (4-MU) generated when the substrate 4-methylumbelliferyl-a-D-glucopyranoside (4-MUGal) was cleaved by AGAL in an acidic pH for 20 h. N-acetyl-D-galactosamine (GalNAc) was used to inhibit alpha-galactosidase B (EC 3.2.1.49) and eliminates its contribution to the measured activity. Results: We studied 88 previously diagnosed Fabry disease patients (73% female) and 690 unaffected controls (41% female). AGAL activity was higher in samples collected using EDTA tubes compared to those spotted directly irrespective of disease status. Conclusions: This assay methodology performs well with high precision, differentiating Fabry from non-Fabry populations. Importantly, this study confirms the need for collection specific reference ranges for alpha galactosidase A, when considering clinical reporting. doi:10.1016/j.ymgme.2008.11.018

18. Fabry disease identification Marsha Browninga, Raphael Schiffmanb, Michael Mauerc, aHarvard/Massachusetts General, Boston, MA, United States, bBaylor Research Institute, Dallas, TX,, cUniversity of Minnesota, Minneapolis, MN Fabry disease is a treatable but clinically under recognized X-linked lysosomal storage disease. High-risk populations, including patients with ischemic stroke and cardiovascular defects, if screened appropriately in a high throughput, cost effective manner, would demonstrate a higher prevalence of Fabry disease than previously reported in restricted patient populations. The prevalence of undiagnosed Fabry in end stage renal disease, including dialysis and kidney transplant recipients, is 20– 50 higher than that of the general population. An aggressive detection platform is needed to identify previously undiagnosed or misdiagnosed patients, including a robust means to detect female Fabry patients, a previously underserved component of these at-risk groups. The specific aims of this project are to: 1. To develop a high-throughput, cost-effective combined biochemical and molecular testing platform for Fabry disease from blood and urine filter paper collection that can be adaptable nationally and internationally for a broader epidemiologic study of detection efficiency. 2. To pilot the most expansive program for Fabry disease detection to date in at-risk populations (stroke and cardiac patients from Texas and Massachusetts; endstage renal disease patients from Minnesota) with the capability of including female patients. 3. To improve diagnostic ascertainment, education, and awareness of Fabry disease. Dr. Browning’s group has developed a modified enzyme analysis using a fluorescence based high throughput microplate (96 well) method as a fully automated system. If scanning is abnormal, full sequence analysis will be performed. Low enzyme levels automatically reflex to full gene sequencing (estimated to be <20% of all samples when tiered through enzyme/scanning processes). Confirmatory molecular testing entails full sequencing of the GLA gene using ABI PRISM 3710 sequencer (Applied Biosystems). A database assembly of mutations and polymorphisms will be maintained. Dr. Schiffmann’s group has recently developed and validated a method for measuring Gb3 using ultra-performance liquid chromatography (UPLC)-MS/MS in whole urine-soaked filter paper. The method uses multiple reaction monitoring positive ion electrospray ionization with a total run of 3 min and results expressed in 50% reduction in subjects to be screened with a <5% loss of identified Fabry patients in order to develop an efficient national end stage renal disease and family screening strategy. 2. Ischemic stroke (3700) patients have been identified through the Harvard Partners RDPR system, and between 3000 and 4000 stroke patients have been identified through the Texas Heart program. All patients with ischemic stroke above 1year-of-age in the stroke clinics through both institution will be offered clinical testing for Fabry disease as a part of routine care. Positive predictive values and prevalence rates will be established linking the biomarkers and genetic mutations via a joint database analysis. 3. Cardiac patients (5800) have been identified through the Harvard Partners RDPR system, and between 8000 and 10,000 stroke patients have been identified through the Texas Heart program for at-risk Fabry conditions, including hypertrophic cardiomyopathy, arrhythmias, unexplained valvular disease, or unexplained coronary artery disease. All cardiac patients at both institutions will be offered

clinical testing for Fabry disease as a part of routine care. Positive predictive values and prevalence rates will be established linking the biomarkers and genetic mutations via a joint database analysis.

doi:10.1016/j.ymgme.2008.11.019

19. Clinical and immunopathologic alterations in Krabbe Rhesus Macaques Bruce Bunnell, Kathrine Phillippi-Falkenstein, Marion Ratterree, Juan Borda, Tulane University School of Medicine, New Orleans, LA, United States Globoid cell leukodystrophy, or Krabbe disease, is a severe metabolic disorder of the central and peripheral nervous system caused by the absence of galactocerebrosidase (GALC) activity. Herein, we describe the clinical, neuropathological, histochemical and immunohistological features observed in rhesus macaques affected with Krabbe’s disease. Clinical signs included pronounced muscle tremors of head and limbs, difficulty ambulating, ataxia, hypermetria, proprioceptive deficits and respiratory abnormalities. Histopathologically, all animals presented with evidence of demyelination in the peripheral and central nervous systems and accumulation of mononuclear and multinuclear globoid cells in the cerebral and cerebellar white matter associated with severe gliosis. Using immunohistochemistry and multi-label confocal microscopy, it was determined that globoid cells were CD68, HAM56, LN5, CD163, IBA-1 and Glut5, suggesting that both peripheral blood-derived monocytes/macrophages and resident parenchymal microglia gave rise to globoid cells. Interestingly, many of the globoid cells and parenchymal microglia with a more ameboid morphology expressed HLA-DR, indicating immune activation. Increased expression of iNOS, TNF-alpha and IL-1 were observed in the affected white matter, colocalizing with globoid cells, activated microglia, and astrocytes. Cytokine mRNA levels revealed markedly increased gene expression of CCL2 in the brain of affected macaques. CCL2-expressing cells were detected throughout the affected white matter, colocalizing with GFAP cells and astrocytes. Collectively, these data suggest that dysregulation of monocyte/macrophage/microglia and up-regulation of specific cytokines may contribute to the pathogenesis of Krabbe disease. doi:10.1016/j.ymgme.2008.11.020

20. Adipose stem cell-mediated therapy for Krabbe disease Bruce Bunnella, Cynthia Ripolla, Mette Flaata, Jeffery Gimbleb, Paul Pistellb, aTulane School of Medicine, New Orleans, LA, United States, bPennington Biomedical Research Center Krabbe disease is a life threatening, inherited glycogen storage disease resulting from mutations in the enzyme galactocerebrosidase (GALC). The only available therapy is heterologous bone marrow transplantation during the earliest months of life to produce the active form of GALC through out the body. Bone marrow transplantation is complicated by graft versus host disease, a severe immunological disorder characterized by engrafted cytotoxic T lymphocytes destruction of host tissues including the skin epithelium, intestinal mucosa, and kidneys. The complications of GVHD extend hospitalizations, increase medical costs, and have a high degree of associated morbidity and mortality. If the clinical diagnosis of Krabbe disease is made later than the first few months, there is no viable treatment option for patients, as bone marrow transplant is no longer effective. Our data indicate that GALC is expressed not only by hematopoietic stem cells but also by adipose derived stem cells. Based on these important observations, we are assessing whether adipose derived stem cells (ASC) have the potential to serve as a cell therapeutic expressing GALC for Krabbe disease without the complicating destructive consequences of hematopoietic stem cell therapy. These studies are focused on the Tiwtcher mouse model of Krabbe disease. Our group is taking a complementary, two-pronged approach to test the ASCs. We are assessing if ASC transplantation into newborn (day 3–4) twitcher mice alters the clinical course of their disorder. ASCs derived from C57Bl/6 mice (syngeneic with twi/twi mice) or Balb/c mice (allogeneic) are being transplanted into the lateral ventricles of twitcher mice. Moreover, we are working to determine if ASCs genetically engineered to over express GALC offer a clinical advantage in the treatment of Krabbe disease symptoms and pathology in twitcher mice. The transduction of ASCs with a lentiviral vector expressing murine GALC (mGALC) cDNA to permit over expression of the enzyme. doi:10.1016/j.ymgme.2008.11.021