Myeloid driven stem cell gene therapy corrects a mouse model of Mucopolysaccharidiosis IIIA

Abstracts

Background: Acid ceramidase (AC) is a lipid hydrolase responsible for the degradation of ceramide to sphingosine. AC activity is deficient in the lysosomal storage disorder, Farber Lipogranulomatosis (FD), due to mutations in the ASAH1 gene. Complete knockout of AC activity leads to early embryonic lethality in mice, highlighting the essential role this enzyme plays in ceramide metabolism and development. Mutations in the AC gene also result in spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), and ceramide accumulation occurs in numerous common diseases as well. Results: Recombinant AC (rAC) was used to modulate ceramide levels in cells and animals with various ceramide-related diseases. For example, addition of the enzyme to cultured FD cells and conditional knockout animals reduced ceramide levels and improved pathology. Addition to in vitro fertilization media enhanced oocyte and embryo survival, and improved birth outcomes after implantation. rAC also improved the production of chondrocytes for cell-based cartilage repair, and was used in vivo to reduce ceramide and prevent Pseudomonas aeruginosa infection in mice with Cystic Fibrosis. Conclusions: Reduction of ceramide using rAC has important therapeutic implications for numerous diseases, including FD, SMA-PME, Cystic Fibrosis and others. The enzyme also may be used ex vivo to enhance cell survival and/or to maintain cell function. doi:10.1016/j.ymgme.2012.11.221

208 Successful screening of 100,000+ newborns for Fabry, Pompe, Mucopolysaccharidosis-I by tandem mass spectroscopy C. Ronald Scotta,b, Susan Elliottc, Norman Burokera,b, Lauren I. Thomasa,b, Michael Glassc, Michael H. Gelba,b, Frantisek Tureceka,b, aUniversity of Washington, Dept. of Pediatrics, Seattle, WA, USA, bUniversity of Washington, Dept. of Chemistry, Seattle, WA, USA, cNewborn Screening Program, Washington State Dept. of Health, Shoreline, WA, USA We have successfully completed a screening program to detect newborns for Fabry disease, Pompe disease, and mucopolysaccharidosis I (MPS I). Over 100,000 anonymous newborns were screened by a multiplex assay that allowed the three enzymes responsible for the disorders to be assayed simultaneously in a single well of a 96-well plate. Quantitation of enzyme activity was performed by a single injection into an MS/MS. Positive screens subsequently received molecular testing to determine disease status. Multiplex screening was performed in the Washington State Newborn Screening Laboratory, with IRB approval. 108,905 infants were screened for alphagalactosidase (Fabry disease). Sixteen samples fell below 18% of the mean, with seven samples confirmed as affected. Prevalence= 1/7,800 males; PPV = 0.44. 111,544 were screened for alpha-glucosidase activity (Pompe disease). Seventeen samples fell below 15% of the mean; four of these samples were consistent with late-onset disease. Prevalence = 1/27,800; PPV = 0.24. The false-positive rate was influenced by a pseudodeficiency allele in the Asian population. 106,526 samples were screened for α-L-iduronidase activity (MPS I). Nine samples fell below 33% of the mean, in which 3 samples were confirmed affected. Prevalence = 1/30,700; PPV = 0.30. In a U.S. population of mixed ethnic background, the combined prevalence of these three LSD is 1/7,700 newborns. The multiplexed assay was successfully integrated into the routine work flow of a newborn screening laboratory. The compelling strength of the MS/MS technology is its ability to quantitate multiple reaction products following a single incubation and a single injection. We anticipate expanding the technology to simultaneously detect additional LSD. doi:10.1016/j.ymgme.2012.11.222

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209 Myeloid driven stem cell gene therapy corrects a mouse model of Mucopolysaccharidiosis IIIA Ana Sergijenkoa, Alexander Langford-Smitha, Ai Yin Liaoa, Claire Pickfordb, John McDermotta, Kia Langford-Smitha, Catherine Merryb, Simon Jonesc, Ed Wraithc, Robert Wynnd, Fiona Wilkinsona, Brian Biggera, a Stem Cell & Neurotherapies, University of Manchester, Manchester, Greater Manchester, UK, bStem Cell Glycobiology, School of Materials, University of Manchester, Manchester, UK, cGenetic Medicine, St Mary's Hospital, Manchester, UK, dBlood and Marrow Transplant Unit, Royal Manchester Children's Hospital, Manchester, UK Mucopolysaccharide (MPS) type IIIA (MPS IIIA, Sanfilippo syndrome type A) is a paediatric lysosomal storage disease caused by mutations in N-sulphoglucosamine sulphohydrolase (SGSH). SGSH deficiency results in heparan sulphate storage and a severe progressive neurodegenerative disease. Enzyme replacement therapy is potentially feasible, but fails because enzyme cannot cross the blood–brain barrier. Haematopoietic stem cell transplantation (HSCT) circumvents this via monocyte trafficking and engraftment in the brain as microglia. HSCT is curative for the similar HS storage disease MPS I but is ineffectual for MPS IIIA which has no treatments. We previously used lentiviral mediated gene therapy to augment SGSH enzyme in WT HSCT, which improves neuropathology and corrects behaviour of MPS IIIA mice. However, correction could not be achieved with lentiviral transduced MPS IIIA cells. To improve expression and brain specificity, lentiviral vectors expressing eGFP under ubiquitous PGK, or myeloid-specific CD11b promoters were compared in transplanted HSC. CD11b gave significantly higher monocyte and B-cell eGFP expression than PGK after 6 months. Subsequently, MPS IIIA HSC were transduced with PGK or CD11b lentiviral vectors expressing codon-optimised SGSH and transplanted into MPS IIIA mice. Eight months after HSCT, PGK lentiviral vectors produced more bone marrow SGSH (576% normal activity) than CD11b (473%), but CD11b was more brain specific (11% vs 7%). CD11b lentiviral vectors fully corrected MPS IIIA behaviour, brain heparan sulphate, GM gangliosides and neuroinflammation to WT levels, whilst PGK lentiviral vectors partly corrected neuropathology but not behaviour. These data provide strong evidence of pre-clinical neurological disease correction using an autologous myeloid-specific lentiviral HSCT approach in the MPS IIIA mouse model. doi:10.1016/j.ymgme.2012.11.223

210 Cognition and neuroimaging in MPS I: Effects of age, severity, and treatment Elsa Shapiroa, Igor Nestrasila, Kyle Rudsera, Kathleen Delaneya, Brianna Yunda, Alia Ahmeda, Kelly Kinga, Victor Kovaca, Julian Raimanb, Paul Harmatzc, Robert Steinerd, Sumar Shankare, Whitley Chestera, aUniversity of Minnesota, Minneapolis, MN, USA, bHospital for Sick Children, Toronto, ON, Canada, cChildren's Hospital & Research Center, Oakland, CA, USA, dOregon Health Sciences University, Portland OR, USA, eEmory University, Atlanta GA, USA We document cognitive and brain imaging abnormalities in mucopolysaccharidosis type I with respect to severity, genotype, treatment, and age. In a multicenter longitudinal study, detailed brain structure and function studies were carried out in MPS I patients. Subgroups classified by severity and by treatment were hypothesized to have different patterns of results. Methods: Seventy-one patients with MPS I were seen at 4 centers were divided into six cohorts: 12 Hurler patients under age 2, seen