altered lysosomal pH) suggesting that storage in this disease is caused byabnormal transport of lipids out of late endosomes or lysosomes. MLIV iscaused by mutations in the MCOLN1 gene encoding the TRP-ML1 puta-tive cation channel believed to be involved in lysosomal calcium and/orproton transport. At present there is no consensus on the exact functionof this channel.

Using highly sensitive microscopy methods, we have measured lyso-somal pH and calcium homeostasis in MLIV fibroblasts. We have foundthat MLIV cells have frequent and continuous spontaneous calciumrelease from lysosomes (whereas normal cells have far fewer lysosomal cal-cium release events). This altered calcium release is associated withincreased late endosome-lysosome fusion and potentially inhibits theirsubsequent reformation. Treatment of MLIV cells with NB-DNJ lowersglycosphingolipid storage and leads to a partial correction of the calciumphenotype, suggesting some involvement of the storage material in exacer-bating the primary underlying phenotype. We propose that the TRP-ML1channel is involved in maintaining normal lysosomal calcium homeostasis,potentially via the NAADP receptor.

doi:10.1016/j.ymgme.2007.10.074

63. Pharmacological chaperone treatment for lysosomal storage disorders

David Lockhart, Amicus Therapeutics, Cranbury, NJ, USA

Lysosomal storage disorders are caused by genetic mutations that leadto a reduction of essential enzymatic activity. In many cases the mutatedforms of the enzymes are functionally competent but less stable and arenot trafficked efficiently from the endoplasmic reticulum to the lysosomes.Pharmacological chaperones are small molecules designed to mitigate thisproblem by specifically binding and stabilizing the target enzyme, thusreducing premature protein degradation and increasing lysosomal enzymeconcentrations and cellular activity. We have developed different pharma-cological chaperones (PCs) for Fabry, Gaucher and Pompe diseases, andall are currently being tested in clinical trials: AT1001 for Fabry in Phase2, AT2101 for Gaucher in Phase 2 and AT2220 for Pompe in Phase 1. Wehave shown that the PCs bind specifically to their target enzyme and thatbinding increases protein stability. The PCs increase cellular enzyme levelsfor multiple mutant forms of each enzyme, as tested using cell lines derivedfrom patients with different mutations and cells transfected with specificmutant forms. The PCs are orally available and well-tolerated in animalsand humans, and they have broad biodistribution into many cell types andtissues. In a Fabry transgenic mouse model that accumulates substrate,AT1001 administration was shown to reduce substrate levels in plasmaand in key tissues such as the heart, skin and brain. These results indicatethat PCs represent a promising new approach and that the specific mole-cules AT1001, AT2101 and AT2220 merit further evaluation as treatmentsfor patients with lysosomal storage disorders.

doi:10.1016/j.ymgme.2007.10.075

64. Targeting neural stem cells in the mammalian brain with intraventricular

injections of lenti, AAV5, or Sleeping Beauty vectors

Walter Low, Terry Burns, Catherine Verfaillie, Perry Hackett, Chet

Whitley, Zhenghong Nan, Feng Xiao, Lalitha Belur, Zach Demorest, Dan

Wolf, John Ohlfest, Scott McIvor, University of Minnesota, Minneapolis,

MN, USA

Neural stem cells (NSCs) in the mammalian brain reside in the subven-tricular zone (SVZ) where they proliferate within this stem cell niche.Upon induction NSCs migrate to other regions of the brain and differen-tiate into neurons and astrocytes. NSCs therefore represent a target forgene therapy that might provide a wide spread distribution to therapeuticcompounds to distant regions of the brain. We have examined the distri-bution of lenti, AAV5, or Sleeping Beauty vectors that express reporter

genes (GFP or luciferase) after intraventricular injections into adult orneonatal mice. Intraventricular injections of lenti-GFP resulted in thelabeling of cells immediately surrounding the ventricles, but few cells wereobserved in the olfactory bulb, the primary destination of migrating stemcells from the SVZ. Intraventricular injections of SB-luciferase alsoresulted in a modest labeling of cells in the subventricular zone. In con-trast, intraventricular injections of AAV5-GFP resulted in a robust label-ing of cells in the SVZ and the olfactory bulb as well as other areas ofbrain. These results suggest that AAV5 appears to exhibit a greater tro-pism for neural stem cells in the SVZ when injected into the lateral ventri-cles in comparison to other vector systems.

doi:10.1016/j.ymgme.2007.10.076

65. Identification of Ambroxol as a potential enzyme enhancement-agent for

Gaucher disease

Don Mahuran a, Gustavo Maegawa a, Michael Tropak a, Justin Butner a,

Gregory Kornhaber b, Brigitte Rigat a, Joe Clarke a, a Hospital For Sick

Children, Toronto, ON, Canada, b ExSAR Corporation, Canada

Gaucher disease, currently treated by enzyme replacement therapy(ERT), is caused by a deficiency of lysosomal beta-glucosidase (GCase).The disadvantages of ERT include its high cost, its ineffectiveness in treat-ing the CNS or other organ compartments and the unfolded proteinresponse in cells. Small molecule-based enzyme enhancement therapy(EET) is a promising approach that can potentially be used alone or incombination with ERT to address these deficiencies. Clinical trials of isof-agomine, an inhibitor of GCase (IC50 � 0.04 lM), as an EET-agent arebeing initiated. In order to accelerate the process of obtaining IND-statusfor new EET-agents we have screened the NINDS library of FDA-approved drugs for compounds that inhibit and/or stabilize the targetenzyme towards heat denaturation. Using GCase as the target we identi-fied Ambroxol, an expectorant, as a candidate EET-agent. DespiteAmbroxol being only a weak inhibitor of GCase, IC50 = 27 lM, at higherconcentrations it compared favorably with isofagomine in its ability to res-cue mutant N370S GCase in patient cells. However, it was not as effectiveat rescuing F213I GCase. Hydrogen–Deuterium exchange Mass Spec-trometry (H/D-Ex) was used to compare the regions in GCase that werestabilized by these compounds. Isofagomine has been shown throughco-crystallization to stabilize a loop structure at GCase352–357. H/D-Exconfirmed these data, while Ambroxol was ineffective at stabilizing thisloop. However, both compounds were equally effective in stabilizinganother region, GCase283–288, indicating its importance as a target forEET-agents.

doi:10.1016/j.ymgme.2007.10.077

66. Development of a disease severity scoring system for patients with

Pompe disease

Deborah Marsden a, Kenneth Berger b, Ans van der Ploeg c, Laura Case d,

Christopher Dandrea a, Priya Kishnani d, Edward Giannini e, a Genzyme

Corporation, Cambridge, MA, USA, b NYU School of Medicine, New

York, NY, USA, c Erasmus MC-Sophia, Rotterdam, The Netherlands,d Duke University Medical Center, Durham, NC, USA, e Cincinatti

Children’s Hospital, Cincinatti, OH, USA

Introduction: A Disease Severity Scoring System (DS3) measures dis-ease burden in patients. It consists of critical health domains, eachdescribed by relevant clinical assessment(s) quantified via reliable, feasiblemethods. DS3s are particularly useful in rare, heterogeneous diseases inwhich evaluating severity and prognosis is difficult. Properly configured,a DS3 provides inter- and intra-patient comparisons through time acrosscritical organ systems. Development is underway for Pompe disease, arare, autosomal recessive, heterogenous, neuromuscular disorder.

S30 Abstracts / Molecular Genetics and Metabolism 93 (2008) S14–S46