The goal of this research is to demonstrate the effectiveness of retroviral-mediated gene transfer therapy for grey collie dogs with cyclic hematopoiesis. Cyclic hematopoiesis is a rare disease that occurs in both man and grey collie dogs and is characterized by cyclical variations in blood neutrophils, monocytes, lymphocytes, eosinophils, reticulocytes and platelets. The recurrent severe neutropenia leads to bacterial infections and shortened life expectancy. Cyclic hematopoiesis can be cured or transferred by bone marrow transplantation, indicating a stem cell defect as the origin of the disease. In affected dogs and humans, long-term administration of recombinant G-CSF produces sustained neutrophilia and abolishes the recurrent hematopoietic cycling. We propose to use vectors encoding G- CSF to infect autologous target cells as vehicles for gene transfer. We will investigate three cell types, vascular smooth muscle cells, skin fibroblasts, and bone marrow cells, chosen for ease of access, culture, transplantability and potential to allow permissive stable expression of transduced gene. We will investigate direct gene transfer by in vivo infection of canine keratinocytes. These studies employ an appropriate canine model of a human hematopoietic disorder. The overall objective of this application is to demonstrate that gene transfer can be used for long-term treatment of this and other genetic diseases.
The specific aims are: (1) Construct selectable retroviral vectors containing canine G-CSF cDNA under the control of various enhancers and promoters. (2a) Infect cultured vascular smooth muscle cells and assay G-CSF expression. Transplant infected/selected autologous cells into dog carotids and veins and monitor persistence and expression of G-CSF. Monitor in vivo effects of transduced gene expression by CBC, cytokine levels and health of animals. (2b) Infect canine skin fibroblasts with these vectors and determine the level of expression and secretion of G-CSF. Transplant infected autologous skin fibroblasts into dogs and monitor their persistence and expression of G-CSF. (2c) Infect canine bone marrow cells in vitro and test for expression of G-CSF in hematopoietic progenitor cells. Transplant infected autologous marrow into dogs and monitor hematopoietic progenitor cells for the expression of G-CSF, and production of peripheral blood cells. (3) In vivo infection of canine keratinocytes using concentrated purified high-titer virus.
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