of chromosome 8. In an ongoing proteomics study of mouse lysosomalmembrane proteins we identified an unknown protein whose humanhomolog, TMEM76, mapped to 8p11.1. A full length mouse ESTwas expressed in human MPS IIIC fibroblasts, and its protein productlocalized to the lysosome and corrected the enzymatic defect. Themouse sequence was used to identify the full length human homolog,which encodes a protein with no homology to other proteins of knownfunction, but is highly conserved through plants and bacteria. Muta-tional analyses of two MPS IIIC cell lines identified a splice junctionmutation which accounted for three mutant alleles, and a single basepair insertion accounted for the fourth.

doi:10.1016/j.ymgme.2007.08.012

8 Sixteen years of experience of biochemical analysis and prenatal diagnosis

of lysosomal storage disease in Iran

Mohammad Hasan Karimi-Nejad, Fariba Afroozan, Bita Bozrgmehr, Navid

Almadani, Valeh Hadavi, Jan G.M. Huijman, Wim J. Kleijer, Otto P. Van

Diggelen, Roxana Kariminejad, Professor of Pathology and Genetics, Karimi-

Nejad-Najmabadi Pathology and Genetic Center, Tehran, Tehran, Iran

Lysosomes are subcellular organelles responsible for the physiologicturnover of cell constituents containing catabolic enzymes requiring alow optimum PH to function. Lysosomal storage diseases (LSD) aredescribed as a heritable group of heterogeneous human disorders charac-terized by the accumulation of undigested macromolecules intra-lysoso-mally, resulting in an increase in the size, number of these organellesand ultimately in cellular dysfunction and clinical manifestations. Lyso-somal storage diseases are generally a broad spectrum and more than 40diseases have been identified so far. Accumulating materials involve allorgans especially central nervous system. Cells of the mononuclear phago-cyte system are rich in lysosomes and so are frequently affected by lyso-somal storage diseases. As the oldest reference center for LSD in Iranwe studied 82 families with 114 members affected with LSD from August1989 to August 2005. Lysosomal storage diseases which were analyzedinclude metachromatic leukodystrophy, Niemann-Pick, Gaucher, Muco-lipidosis, Canavan, Alexander, GM1-Gangliosidoses, GM2-Gangliosidos-es (Tay-Sachs and Sandhoff), Fabry disease, Krabbe disease, Fucosidosis,Wolman_s disease, Neuronal Ceroid lipofuscinosis, Sea blue histiocytosis,Lipodiosis, Pyknodysostosis, Cystinosis, sialidosis.

We performed a total of 36 prenatal diagnoses from 77 couples at riskfor LSD. Biochemical assays were applied for all cases. Twenty threefetuses (64%) were found to be normal, and 12 (34.4%) were affected.The test results of one of the samples are not ready yet. Our data supportsthe functionality of biochemical analysis of course when the families withlive affected child referred to the diagnostic centers before losing theiraffected child.

doi:10.1016/j.ymgme.2007.08.013

9 Utilizing the brain microvasculature for CNS therapy

Beverly Davidson a, Yong-Hong Chen b, a University of Iowa College of

Medicine, Iowa City, IA, USA, b Departments of Internal Medicine,

Neurology and Physiology & Biophysics, University of Iowa, Iowa City, IA,

USA

Adeno-associated viruses (AAV) are attractive vectors for genereplacement therapy because they have limited toxicity and express trans-genes for long time periods. Collectively, the different serotypes of AAVshow broad tissue and cell tropism. However, AAV does not efficientlytransduce endothelia when administered in vivo. In our approaches todevelop gene therapy for the CNS manifestations of lysosomal storage dis-eases (for example, the mucopolysaccharidoses and the neuronal ceroidlipofuscinoses), endothelia remain an attractive target. Lysosomal

enzymes deficient in these disorders, when expressed from endotheliain vitro, are apically and basolaterally secreted; in the brain, this wouldallow exposure of recombinant enzyme to the underlying brain paren-chyma. To develop brain-endothelia targeting vectors, we first used phagedisplay to identify peptides that mediate selective and efficient binding tobrain endothelial cells in vivo. Mice deficient in b-glucuronidase and wild-type mice were used for panning. Interestingly, distinct epitopes werepanned, suggesting vascular remodeling in this disease model. Selectedpeptides panned from the b-glucuronidase deficient mice were geneticallyincorporated into AAV2 capsids, and peptide modified viruses(PM-AAVs) generated and tested in vitro and in vivo. We found theAAV2-ITR genomes were efficiently packaged into the novel capsids, andPM-AAVs retained brain targeting after intravascular delivery. We nexttested the therapeutic efficacy of the PM-AAVs in b-glucuronidase-deficientmice. Following intravenous delivery of vector via tail vein, there was exten-sive expression of enzyme in brain, correction of lysosomal storage, andimportantly, reversal of established behavioral deficits. These studies arean important first step towards accomplishing CNS therapy after peripheraldelivery for this group of fatal, childhood onset inborn errors.

doi:10.1016/j.ymgme.2007.08.014

10 Neonatal gene therapy reduces clinical manifestations in MPS I dogs

Anne M. Traas, Ping Wang, Xiucui Ma, Patricia O_Donnell, Meg Sleeper,

Gus Aguirre, Mark Haskins, Katherine Parker Ponder, Washington

University School of Medicine, St. Louis, MO, USA

Mucopolysaccharidosis I (MPS I) due to deficient activity of a-L-idu-ronidase (IDUA) results in bone, joint, eye, heart, and neurological dis-ease. The MPS I dog has a 5_ splice site mutation in intron 1 anddisease that resembles that in humans. As previous attempts at hematopoi-etic stem cell-directed gene therapy have failed due to an immuneresponse, the dog MPS I may be an excellent model for immune responsesin patients with null mutations. Six MPS I dogs were treated at three daysof age with intravenous injection of 3–10 · 10E9 transducing units(TU)/kg of a retroviral vector (RV) expressing canine IDUA from thehuman 1-antitrypsin promoter (hAAT-cIDUA-WPRE). This resulted in22–749 U/ml of enzyme activity in serum (1.3- to 44-fold normal), whichhas been stable for up to 21 months. RV-treated dogs had reduced bio-chemical evidence of lysosomal storage in liver, spleen, kidney, and brain.Clinical manifestations in untreated and RV-treated dogs were scoredfrom 0 (normal) to 3 (severe). At 12 months of age, untreated MPS dogshad facial dysmorphia (score = 3.0000), corneal clouding (score = 2000),mitral valve thickening (score = 1.5000.6), and mild aortic dilatation(score = 1000.8). Scores for RV-treated dogs at 1 year of age were 0 forfacial dysmorphia, 0.6000.5 for corneal clouding, 0.3000.0 for mitral valvethickening, and 0000 for aortic dilatation. We conclude that neonatal genetherapy is effective at correcting the clinical manifestations of MPS I indogs.

doi:10.1016/j.ymgme.2007.08.015

11 Adeno-associated virus vector-mediated gene therapy in a murine model

of mucopolysaccharidosis type I

Lalitha Belur a,b, Zachary deMorest c, Chester B. Whitley a,d, Walter C.

Low c, R. Scott McIvor a,b, a Gene Therapy Program, Institute of Human

Genetics, University of Minnesota, Minneapolis, MN, USA, b Department of

Genetics, Cell Biology and Development, University of Minnesota, Minneapolis,

MN, USA, c Department of Neurosurgery, University of Minnesota, Minneapolis,

MN, USA, d Department of Pediatrics, University of Minnesota, Minneapolis,

MN, USA

Adeno associated virus (AAV) is a replication deficient human parvo-virus, with a single stranded DNA genome and no known human

Abstracts / Molecular Genetics and Metabolism 92 (2007) S11–S34 S13