Gene replacement in mutant marrow cells is a potential treatment modality for heritable disorders of the blood forming tissues and for specific metabolic diseases. A major problem with bone marrow transfer therapy is our incomplete understanding of the injected stem cells' fate. This is critical following virus mediated gene therapy where active clones appear to be lost or suppressed. While the mouse is an excellent model in which to assess stem cell biology, events occurring after marrow transplantation have, of necessity, been assayed not in situ but either in vitro or in a secondary host. We have, until recently, lacked a good model in which host and donor cells are distinguishable. We are studying a mouse model of the human lysosomal storage disease Mucopolysaccharidosis Type VII, MPS VII, that lends itself to the direct in vivo analysis of normal and virus transduced MPS VII stem cells. The MPS VII mice lack the enzyme beta- glucuronidase but all +/+ donor cells (except erythrocytes) and MPS VII cells that are transduced to produce beta-glucuronidase are recognized by their intense histochemical staining against a null background. Our goals are to understand the fate of the injected cells, to assess cell lineages derived from them, and to examine virus transduced cells for continuing beta-glucuronidase production.
The Specific Aims are: To establish the sites where injected +/+ hematopoietic cells either from whole marrow or from marrow highly enriched for stem cells seed in the MPS VII recipient; To identify in vivo the immediate progeny of the cells in Aim 1; To assess the long term distribution of the donor progeny in peripheral blood and various organs of the beta-glucuronidase deficient mice; To determine the relative numbers of donor autologous MPS VII stem cells that are transduced by exposure to virus carrying the rat beta- glucuronidase cDNA and to assess their production of beta-glucuronidase in vivo; and To monitor the long term production of beta-glucuronidase in virus transduced cells in the various MPS VII tissues. Results address hypotheses concerning seeding site preference, inter-site migration, position-and time-dependent differentiation, lineage complexity, and long term function of transduced genes, impossible to assess directly until now.
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