The lysosomal storage diseases are inherited metabolic deficiencies that produce fatal degenerative syndromes, many of which include mental retardation. Evidence from bone marrow transplantation and transgenic studies indicates that supplying the normal gene product early in life may significantly reduce pathology compared to treating older patients. We will develop methods to transfer genes to the developing fetus in utero as an approach to early treatment that may reduce the severity, or prevent the development, of pathology. Mice and dogs with beta-glucuronidase (GUSB) deficiency, which results in mucopolysaccharidosis (MPS) VII (Sly disease), will be used as model systems. We maintain breeding colonies of carrier mice and dogs, and have developed retroviral vectors to transfer and express high levels of GUSB in MPS VII cells. The experimentally treated animals will bc evaluated after birth for the presence of the transferred gene, the distribution of transduced cells, expression of the transferred gene, the distribution of exported enzyme, alterations in pathology, and changes in the clinical syndrome. We will transfer genes into the fetus by infecting fetal liver cells (hematopoietic precursors) ex vivo and transplanting them into the fetus in utero. We will follow the fate of the donor cells by determining the distribution of their progeny using the vector proviruses as genetic markers. We will investigate the expression and activity of the transferred GUSB gene in the MPS VII animals by using a cytochemical reaction to directly assay for the presence of GUSB enzymatic activity in situ against the negative background of the MPS VII tissues. The long-term effects on pathology will be evaluated, especially in the brain, because the blood-brain barrier is a formidable obstacle to efforts to deliver GUSB to the brain in adult MPS VII animals. During development, evidence suggests that fetal hematopoietic cells may migrate from the liver to the brain late in gestation and give rise to the microglia. Our experiments will determine whether vector-infected donor cells colonize brain. We will also determine the extent to which fetal cells can be transduced in situ by direct injection of vector virus. In these experiments we will determine the effectiveness of correcting cells that are refractory to vector transduction postnatally, e.g. neurons, but which can be infected while they are undergoing mitosis during development. These transduced fetal cells may give rise, through normal growth, to tissues that contain larger numbers of corrected cells than can be achieved by gene transfer to more fully developed tissues.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK046637-01
Application #
3248046
Study Section
Medical Biochemistry Study Section (MEDB)
Project Start
1993-05-01
Project End
1996-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Veterinary Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Walton, Raquel M; Wolfe, John H (2008) In vitro growth and differentiation of canine olfactory bulb-derived neural progenitor cells under variable culture conditions. J Neurosci Methods 169:158-67
Magnitsky, Sergey; Walton, Raquel M; Wolfe, John H et al. (2008) Magnetic resonance imaging detects differences in migration between primary and immortalized neural stem cells. Acad Radiol 15:1269-81
Walton, Raquel M; Wolfe, John H (2007) Abnormalities in neural progenitor cells in a dog model of lysosomal storage disease. J Neuropathol Exp Neurol 66:760-9
Magnitsky, S; Walton, R M; Wolfe, J H et al. (2007) Magnetic resonance imaging as a tool for monitoring stem cell migration. Neurodegener Dis 4:314-21
Cearley, Cassia N; Wolfe, John H (2006) Transduction characteristics of adeno-associated virus vectors expressing cap serotypes 7, 8, 9, and Rh10 in the mouse brain. Mol Ther 13:528-37
Karolewski, Brian A; Wolfe, John H (2006) Genetic correction of the fetal brain increases the lifespan of mice with the severe multisystemic disease mucopolysaccharidosis type VII. Mol Ther 14:14-24
Watson, Deborah J; Walton, Raquel M; Magnitsky, Sergey G et al. (2006) Structure-specific patterns of neural stem cell engraftment after transplantation in the adult mouse brain. Hum Gene Ther 17:693-704
Watson, Deborah J; Passini, Marco A; Wolfe, John H (2005) Transduction of the choroid plexus and ependyma in neonatal mouse brain by vesicular stomatitis virus glycoprotein-pseudotyped lentivirus and adeno-associated virus type 5 vectors. Hum Gene Ther 16:49-56
Jiang, Kanli; Watson, Deborah J; Wolfe, John H (2005) A genetic fusion construct between the tetanus toxin C fragment and the lysosomal acid hydrolase beta-glucuronidase expresses a bifunctional protein with enhanced secretion and neuronal uptake. J Neurochem 93:1334-44
Magnitsky, S; Watson, D J; Walton, R M et al. (2005) In vivo and ex vivo MRI detection of localized and disseminated neural stem cell grafts in the mouse brain. Neuroimage 26:744-54

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