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.
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