138BMN 701-mediated receptor redistribution is responsible forincreased uptake

Jonathan H. LeBowitza, John Magaa, Ben Schoolera, Ginger Chena,Erno Pungora, Bill Princea, Gangzhi Liub, Yuanfeng Xiab, aBioMarin,Novato, CA, USA, bHD BioSciences, Shanghai, China

BMN 701 is a novel chimeric fusion protein of Insulin-like GrowthFactor 2 (IGF-2) and acid alpha-glucosidase (GAA) that is now beingtested in the clinic for the treatment of Pompe disease. BMN 701 wasdesigned to improve delivery of GAA to the lysosome of muscle cells byfusing a high affinity ligand for the cation independent mannose-6-phosphate receptor (CI-MPR) to GAA. In uptake experiments with rat L6myoblasts, the Kuptake improvedmarkedly compared to untagged rhGAA,a predicted consequence of higher affinity binding to the CI-MPR.Additionally, the total amount of BMN 701 taken up by cells (systemcapacity) was doubled compared to rhGAA. This surprising observationcould be explained by a phenomenon reported previously, namely thatIGF-1 and IGF-2 can induce a signaling cascade resulting in redistributionof the CI-MPR so as to increase the quantity of the CI-MPR on the cellsurface. We have investigated the ability of BMN 701 to do the same. Invitro, we find: i) BMN 701 can bind to the IGF-1 receptor; ii) BMN 701can transduce a signaling cascade from the IGF-1 receptor; iii) theelevated system capacity of BMN701 is dependent on this signaling fromthe IGF-1 receptor; and iv) system capacity for uptake of untaggedrhGAA can be increased by IGF-1. These results are consistent with thehigher system capacity resulting from tag-dependent signaling. Thisnovel attribute of BMN 701 may contribute to its enhanced ability toreverse glycogen storage in Pompe mice.

doi:10.1016/j.ymgme.2013.12.150

139Prediction of the molecular consequences of aminoacidsubstitutions in the GALNS gene using in silico tools

Sandra Leistner-Segal, Francyne Kubaski, Ana Carolina Brusius-Facchin, Aline Nemetz Bochernitsan, Roberto Giugliani, Hospital deClínicas de Porto Alegre, Porto Alegre/RS, Brazil

Computational methods are used to predict the molecular conse-quences of amino-acid substitutions on the basis of evolutionaryconservation or protein structure, being relevant tools for the establish-ment of the genotype-phenotype correlation, particularly for novelmutations in molecular heterogeneous diseases, such as Morquiosyndrome type A (MPS IVA). MPS IVA is an autosomal recessive inbornerror of metabolism caused by deficiency of lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). To date, almost 200different mutations associated with MPS IVA have been identified. Theprediction of 3 novel missense mutations p.V16E, p.E51K and p.C165Y,found in the GALNS gene, were analyzed using four different programs:PANTHER (based on evidence of evolutionary conservation of amino-acids and identification of amino acid positions that are known to beessential for protein function, SIFT (uses sequence homology), PolyPhen2 (offers predictions based on conservation, protein folding and crystalstructure) and PMUT (retrieves information from a database of hotspotmutations and analyze SNPs in a specific protein). The PANTHER 8.1scores range from 0 (no effect) to negative values; SIFT ranges from 0(most deleterious) to 1.0 (completely tolerable change); Polyphen 2ranges from benign (no effect), possibly damaging and probablydamaging; PMUT (pathological or neutral). The results obtained afterin silico analysis were: PANTHER 8.1(p.V16E -2.78; p.E51K -2.25;p.C165Y -4.05), SIFT (p.V16E: 12:26 tolerated; p.E51K: 0.5 tolerated;

p.C165Y: 0:03 deleterious), Polyphen 2(p.V16E: 0718 possibly deleteri-ous; p.E51K: 0849 possibly deleterious; p.C165Y: 1 probably deleteri-ous), PMUT (p.V16E neutral; p.E51K neutral, p.C165Y pathological). Theidentification of novel mutations in MPS IVA and prediction of thephenotype, are challenges in molecular diagnosis of this syndrome. Thenovel mutations described here can be classified based on clinicalfeatures as having attenuated phenotype (p.V16E, p.E51K) and severephenotype (p.C165Y). All software used showed agreement in definingthe severity of themutations, and can be considered good tools to clarifythe genotype-phenotype correlation. Acknowledgements: FIPE-HCPA,INAGEMP, CNPq.

doi:10.1016/j.ymgme.2013.12.151

140Mitochondial abnormalities in Pompe disease

Jeong-A Lim, Nina Raben, NIAMS/NIH, Bethesda, MD, USA

Pompe disease, glycogen storage disease type II, is a rare geneticdisorder caused by a defect of lysosomal enzyme, acid α-glucosidase(GAA), which breaks down glycogen to glucose. Clinically, the diseasemanifests as severe cardiac and skeletal muscle myopathy. Enzymereplacement therapy with recombinant human GAA has been successfulin reversing cardiac but not skeletal muscle pathology. Our recent dataprovide strong evidence that skeletal muscle resistance to therapy isassociated with the presence of large pools of autophagic debris in thistissue in both our GAA knockout (KO) model and in patients affectedwith Pompe disease. Defective autophagy is expected to lead tomitochondrial abnormalities since anomalous mitochondria are elimi-nated by the autophagic pathway (a process called mitophagy). Indeed,morphologically abnormal mitochondria sequestered in autophagicvesicles which are unable to reach the recycling place, the lysosomes,have been sighted in muscle biopsies of Pompe patients and KO mice.Here, we analyzed the functional status of mitochondria in the KO miceand in a recently generated cell model of Pompe disease. The geneexpression profiles generated from KO muscle revealed a strikingupregulation of L-type Ca2+ channel isoforms (an increase of hundredtimes for some of the subunits) compared with WT; the data wereconfirmed by western analysis in both in vivo and in vitro models. As aresult, intracellular Ca2+ concentration (detected in live cells by usingcalcium fluorescent dye) and Ca2+ flux (shown by time-lapse micros-copy) were greatly increased in KOmuscle cells. These data indicate thatin the diseased muscle there is a profound dysregulation of Ca2+

homeostasis - a key regulator of mitochondrial function. The geneexpression studies also showed a modest but significant upregulation ofmitochondrial Ca2+ uniporter and downregulation of Na+/Ca2+

exchanger which are responsible for the uptake and extrusion of Ca2+

by mitochondria, respectively. The intracellular and mitochondrial Ca2+

overload were associated with ROS (reactive oxygen species) increaseand a greater vulnerability to hydrogen peroxide-induced oxidativestress in KO muscle cells compared with WT. Next, we demonstratedthat altered intracellular Ca2+ concentration provoked a decrease inmitochondrial membrane potential and a release of apoptosis-inducingfactor (AIF) in KO muscle cells. The impaired mitochondrial function inPompe muscle was further supported by the finding of a decreasedoxygen consumption of mitochondria isolated fromwhole muscle of theKOmice. Finally, nifedepine - a Ca2+channel blocker - partially reversedoxidative stress-induced abnormalities. Taken together, the data uncoveryet another pathogenetic mechanism leading to muscle damage inPompe disease and point to new targets for therapeutic intervention.

doi:10.1016/j.ymgme.2013.12.152

Abstracts S67