agy exerts a pro-survival effect in the NPC brain, and discuss potential therapeuticstrategies for modulating activity of this pathway.

doi:10.1016/j.ymgme.2008.11.082

82. Substrate reduction therapy in Niemann-Pick disease type C: Report of fiveBrazilian patients

Charles Lourencoa, J.S. Camelo Jr.a, M.L.S.F. Santosb, V. Van Der Lindenc, M. Turcatoa,W. Marques Jr.a, aClinics HospitalUniversity of Sao Paulo, Ribeirao Preto, Sao Paulo,Ribeirao Preto, Sao Paulo, Brazil, bPequeno Principe Hospital, Curitiba, Parana, cAACD,Recife, Pernambuco

Background: In Niemann-Pick disease types A/B, lipid, mainly sphingomyelin,accumulates; in Niemann-Pick type C (NPC), there is, instead, a defect in cholesterolesterification causing lipid storage.

Methods: Five patients with clinical, biochemical and/or molecular diagnosis ofNPC were evaluated with a clinical protocol/physical examination and complemen-tary exams (abdominal ultrasound, brain MRI,EEG,NCV) during one year of treatmentwith the imino sugar N-butyldeoxynojirimycin (NB-DNJ).

Results: Four patients but one were confirmed to have NPC by Filipin test (one pa-tient required molecular analysis). Regarding clinical form, 1 was perinatal, 2 wereinfantile and 2 juvenile ( ages varying from 12 months to 13 years). Four patientshad started on an inhibitor of glycosphingolipid storage (NB_DNJ) after the onset ofthe neurological symptons. The youngest patient12 months old had started on themedication because of severe hepatosplenomegaly of perinatal onset. All patientshave showed regression of hepatomegaly, although various degree of splenomegalystill remain. Stabilization of mental deterioration was seen in the patients with neu-rological symptons one of the patients with the juvenile form had remarkableimprovement of neurological features lder patient. The youngest patient, despite ofthe treatment, presented with seizures with 14 months. However after one year withthe treatment, she does not have seizures anymore, her EEG normalized and she be-gan to walk with 2 years old. Electrophysiological studies did not demonstrate anyperipheral neuropathy. Plasma chitotriosidase activities were stabilized in all cases;only one patient had decrease of chitotriosidase after one year of treatment. Electro-physiological studies did not demonstrate any peripheral neuropathy.

Conclusions: Substrate reduction therapy seems to be a reasonable approach totreat lysosomal storage disorders, specially those disorders with brain involvement.Nevertheless, it is necessary to develop better biomarkers to follow the treatment re-sponse in such patients.

doi:10.1016/j.ymgme.2008.11.083

83. The effects of altering calcium homeostasis on mutant enzyme activity inchronic Tay-Sachs disease and Gaucher disease patient cells treated withpharmacological chaperones

Don Mahuran, Brigitte Rigat, The Hospital For Sick Children, Toronto, ON, Canada

Recently, inhibition of L-type Ca2+ channels, using either diltiazem or verapa-mil, has been reported to partially restore mutant glucocerebrosidase (Gcc) activityin cells from patients with Gaucher disease (GD) homozygous for the N370S orL444P alleles, as well as cells from patients with two other lysosomal storage dis-eases. It was hypothesized that these drugs act by increasing the folding efficiencyof the endoplasmic reticulum-quality control system, inhibited due to altered cal-cium homeostasis. It is significant that cells carrying either the N370S or the F213Ialleles are amenable to enzyme enhancement therapy with pharmacological chap-erones (PCs), whereas L444P homozygous cells have shown little response to PC-treatment. We found that verapamil produces little or no enhancement of activityin any of the cell lines tested, while diltiazem moderately increases enzyme activ-ity in N370S/N370S and F213I/L444P GD cells, but not in L444P/L444P GD cells orin either of two adult Tay-Sachs disease (ATSD) cell lines. Since the mode of actionof PCs and diltiazem are believed to be different, we examined the possibility thatPCs and diltiazem could act in concert to further enhance mutant enzyme activityin either GD or ATSD cells. Diltiazem co-administered with known PCs (isofago-mine, GD; pyrimethamine, ATSD) failed to increase enzyme activities above thatreached by PC-treatment alone in any of the patient cell lines. Interestingly, inF213I/L444P GD cells diltiazem inhibited the enhancement of Gcc activity pro-duced by isofagomine. We conclude that: (1) diltiazem may be acting through apathway distinct from its ability to inhibit L-type Ca2+ channels; (2) the rangeof LSDs amenable to its treatment may be limited; and (3) co-treatment withPCs will likely produce no added efficacy.

doi:10.1016/j.ymgme.2008.11.084

84. Adeno-associated virus gene therapy of feline gangliosidosis

Douglas Martina, M. Begona Cachon-Gonzalezb, Miguel Sena-Estevesc, Rena C. Baekd,Thomas N. Seyfriedd, Timothy M. Coxb, Nancy R. Coxa, aAuburn University, AL, USA,bUniversity of Cambridge, AL, USA, cMassachusetts General Hospital, AL, USA, dBostonCollege, AL, USA

AAV gene therapy has been extraordinarily successful in mouse models of GM1and GM2 gangliosidosis (Mol. Ther., 15 (2007) 30; Proc. Natl. Acad. Sci. USA, 103(2006) 10373). Because the mouse brain is less complex and 1000 times smaller thanthe human brain, AAV gene therapy must be tested in large animals before beginninghuman clinical trials. When AAV vectors of serotype 1, 2, 8, 9 or rh10 were injectedinto the cat thalamus, enzymatic activity was detected 1.4–2.0 cm from the injectionsite. AAV1 or AAVrh10 vectors were superior to AAV2 or AAV8, which produced focalareas of enzymatic activity <0.2 cm from the injection site. Initial therapeutic studiesin GM1 cats (beta-galactosidase deficient) consisted of bilateral thalamic injections ofAAV1 vector (1.2–1.5 10(12) g.e.) with or without prior IV injection of an AAV8 vectorcontaining a liver-specific promoter (1.0–1.5 10(13) g.e./kg). Brain enzymatic activityranged from 54.9–296.9% of normal over a distance of 1.5 cm, and a GM1 cat treatedby intrathalamic injection alone lived to 8.9 months. Two GM1 cats treated by IV/intrathalamic injections lived to 9.6 and 10.0 months of age (untreated GM1 life spanis 7.7 0.8 months, n = 9). Similar results were obtained in GM2 cats (hexosaminidasebeta-subunit deficient) injected intrathalamically with AAV1 vectors: (a) specificactivity ranged up to 347.8% and 18.7% of normal in the injected and contralateralhemispheres, respectively; (b) sialic acid levels were reduced by up to 2/3; (c) GM2storage was substantially but not completely cleared at the injection site; (d) GM2cats treated by intrathalamic injection with or without IV pretreatment lived to 8.0or 7.0 months, respectively (untreated GM2 life span is 4.5 0.5 months, n = 11). Genetherapy results in mice and cats strongly support the continued development of AAVvectors for human gangliosidosis clinical trials.

doi:10.1016/j.ymgme.2008.11.085

85. Natural history and structural–functional relationships in Fabry renal disease

Michael Mauer, Behzad Najafian, Basgen John, University of Minnesota, Minneapolis,MN, USA

Chronic renal failure is a major debilitating and life-threatening complication ofFabry disease known to account for 0.01% of end-stage renal disease in western coun-tries. Enzyme screening studies in male dialysis patients suggest that this might be anunderestimate of the true prevalence of the disease by 10- to 100-fold. Furthermore,the natural history of heterozygous females is even more unclear. Primary prevention,before the disease is clinically manifest, could be important to maximize the potentialbenefits of enzyme replacement therapy, but it will be important to evaluate suchearly intervention before consigning every child to life-long therapy. Understandingthe cellular pathology, and the tempo of progression, will be crucial. However, thestructural changes of Fabry renal disease responsible for glomular filtration rate(GFR) loss have not been systematically studied. Using quantitative morphometricstereologic methods we have recently demonstrated that podocyte GL-3 accumula-tion is progressive with age (time) while mesangial and endothelial cell accumulationis not. We will apply these systematic quantitative methods to 50–60 Fabry patientswith a wide range of GFR with kidney biopsies performed prior to beginning enzymereplacement therapy. We will then develop a model of structural functional relation-ships which most closely predicts GFR loss. These data will be used for the power cal-culations needed to design early intervention trials based on those structuralendpoints which are most closely related to important functional outcomes in Fabrydisease.

doi:10.1016/j.ymgme.2008.11.086

86. Ex vivo lentiviral gene transfer targeting hematopoietic stem cells for therapyof mucopolysaccharidosis type II, Hunter syndrome

R. Scott McIvora, Daniel A. Wolfa, Kelly Podetz-Petersena, Zhenhong Nana, Chester B.Whitleya, Joseph Muenzerb, Paul J. Orcharda, Walter C. Lowa, aUniversity of Minnesota,Minneapolis MN, Minn, MN, USA, bUniversity of North Carolina, Chapel Hill NC

The mucopolysaccharidoses (MPS) are lysosomal storage diseases caused by defi-ciency of one of the enzymes in the pathway for breakdown of glycosaminoglycans(GAG). The MPS are considered excellent candidate conditions for gene therapybecause cellular complementation studies have shown that the enzymes of GAGmetabolism can be taken up by genetically deficient cells and trafficked to lysosomes.This process, termed metabolic cross-correction, is the mechanistic basis for the effec-tiveness of enzyme replacement therapy (ERT) and allogeneic hematopoietic stem cell

S30 Abstracts / Molecular Genetics and Metabolism 96 (2009) S12–S47