INBORN ERRORS OF METABOLISM: LYSOSOMAL STORAGE AND OTHER 697. Correction of Lysosomal Storage within the Brain of Adult MPS VII Mice Following Intrahepatic Administration of a Recombinant Adeno-Associated Virus Vector 1
Kristin Backstrom, Chuansong Wang, Chiou-Miin Wang, Thomas J. Sferra.1 1 Center for Gene Therapy, Columbus Children’s Research Institute, Columbus, OH.
Mucopolysaccharidosis type VII (MPS VII; Sly disease) is a lysosomal storage disease (LSD) caused by a deficiency of the acid hydrolase β-glucuronidase. Deficiency of this enzyme results in the lysosomal accumulation of glycosaminoglycans in most organs, including the brain. In the mouse model of this disorder, widespread correction of the central nervous system manifestations has not been observed with peripherally administered therapies in adult animals. The objective of this study was to evaluate the effect of peripheral administration of an rAAV vector on lysosomal storage within the brain of adult MPS VII mice. Methods: An rAAV type 2 vector (rAAV-Gus.2.1) carrying the murine β-glucuronidase (Gus) cDNA under the transcriptional direction of the human elongation factor-1α promoter was administered to adult (6-8 weeks of age) MPS VII mice. Each mouse received 6 x 1011 DNase resistant particles by direct intrahepatic injection. Groups of mice were sacrificed at 4, 12, and 24 weeks post-vector administration. The brains were cut into 2-mm coronal sections. Specific anatomic regions were dissected from each section, embedded in plastic, sectioned, and stained with toluidine blue. An investigator blinded to the treatment groups scored the degree of lysosomal storage (cytoplasmic vacuolation) within the brain. βglucuronidase activity (fluorometric assay) and vector genome levels (quantitative polymerase chain reaction assay) were determined in mice sacrificed at 12 weeks post-injection. Results: The degree of lysosomal storage within the brains of mice 4 weeks post-vector administration (n = 3) was no different from age-matched control mice. At 12 and 24 weeks (n = 3 mice per time), reductions in lysosomal storage were found in all areas examined, including the olfactory bulb, cortex (motor, somatosensory, and piriform), striatum, corpus callosum, hippocampus, and cerebellum. The degree of improvement was progressive over time. At 24 weeks, the majority of regions examined were no different from normal mice. High levels of storage were observed only within the Purkinje cells of the cerebellum and low levels within pericytes scattered throughout the brain. At 12 weeks post-vector injection (n = 3 mice), β-glucuronidase activity within the liver was 1050±229%, serum 226±15%, and brain 0.7±0.1% of normal, wild-type levels. Vector genome levels were 8.0±5.3 and 0.013±0.002 vector genomes per cell genome equivalent for the liver and brain, respectively. Summary: These data demonstrate that correction of the lysosomal storage abnormality within the brain of adult MPS VII mice is possible following the peripheral administration of an rAAV vector. Further studies are necessary to determine the mechanism by which disease correction occurs. These results have significant implications in the potential use of gene transfer for treatment of this and other neuronopathic LSDs. Supported by NIH, NINDS R01NS39071.
Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright The American Society of Gene Therapy
698. Gene Therapy of Murine GM1 Gangliosidosis by Genetically Modified Bone Marrow Hematopoietic Progenitor Cells Renata Sano,1 Alessandra Tessitore,1 Angela Ingrassia,1 Alessandra d‘Azzo.1 1 Department of Genetics and Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, TN. b-galactosidase (b-gal), a lysosomal enzyme involved in the removal of b-linked terminal galactosyl residues of many glycoconjugates, is deficient in the neurodegenerative lysosomal disorder GM1-gangliosidosis (GM1). GM1-/- mice closely mimic the most fundamental aspects of the neuropathological and neurochemical abnormalities of the human disorder. Bone marrow progenitor cells have been used as a source of corrective protein because of their ability to repopulate the recipients and to supply functional enzyme to different cells by “in trans” correction. Hematopoietic progenitors transduced with a murine stem cell virus (MSCV)-based bicistronic retroviral vector over-expressing b-gal and the green fluorescent protein (GFP) marker were used for transplantation into sublethally irradiated GM1-/- mice. Transduction efficiency of total BM cells with the MSCV-b-gal ranged from 25-89% prior to the transplantation. b-gal expressing BM-derived cells were detected histologically and enzymatically in many tissues including spleen, liver, lungs, intestine and kidney after one, three and six months post transplantation (BMT). GFPexpressing cells of the erythroid, myeloid or lymphoid lineage were identified by FACS analysis of peripheral blood samples, collected at different time points after transplantation. Secondary transplantations demonstrated consistent long-term “in vivo” b-gal expression in all tissues including brain. In addition, the thin layer chromatography of brain lipids showed a reduction in the GM1ganglioside content in brainstem and cerebellum after three and six months post BMT. In line with the biochemical and histological findings, different behavioral tests including open field, rotorod and walking pattern, indicated a clear improvement of the neurological function in transplanted GM1 -/- compared to untreated littermates. Taken together these results have encouraged the use of ex-vivo gene therapy for the treatment of GM1 (Supported in part by NIH grant DK 52025).
699. Correction of Glycogen Storage Disease Type II (GSD II) with an Adeno-Associated Virus 8 (AAV2/8) Vector Baodong Sun,1 Haoyue Zhang,1 Luis Franco,1 Ayn Schneider,1 Andrew Bird,1 Andrea Amalfitano,1 Y.-T. Chen,1 Dwight D. Koeberl.1 1 Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, NC. Glycogen storage disease type II (GSD II; Pompe disease; MIM 232300) causes death in infancy from cardiomyopathy and cardiorespiratory failure. The underlying deficiency of acid alphaglucosidase (GAA; acid maltase; EC 18.104.22.168) has been corrected by enzyme replacement in animal models and in clinical trials. Gene therapy could provide long-term, beneficial replacement of GAA in GSD II, if a depot organ secreted GAA accompanied by receptormediated uptake in skeletal and cardiac muscle. Intravenous administration of adenovirus vectors encoding GAA previously demonstrated generalized correction of glycogen storage in the GAAknockout (GAA-KO) mouse model, although glycogen gradually re-accumulated in the months following vector administration. We administered AAV vectors encoding human GAA in GAA-KO mice, and subesquently analyzed GAA activity and glycogen content in tissues. GAA was delivered to skeletal muscle and heart following portal vein injection of an AAV2/2 vector (1 x 10E12 particles) in S265