The goal of this research is to demonstrate the effectiveness of retroviral-mediated gene transfer as therapy for grey collie dogs with cyclic hematopoiesis. Long-term administration of recombinant canine G-CSF abolishes the recurrent hematopoietic cycling in affected dogs. Dr. Osborne and his colleagues propose to use vectors encoding canine G-CSF cDNA to transduce autologous target cells as vehicles for gene transfer and to infect target cells in vivo. They will investigate implantation of vascular smooth muscle cells in prosthetic PTFE grafts and PTFE stomach implants to provide safe long-term expression of G-CSF. Skin fibroblasts will be transplanted as skin equivalent grafts using elements of rat fibronectin promoter to express G-CSF and overcome retroviral vector inactivation. These target cells were chosen for ease of access, culture, transplantability and potential to allow stable expression of transduced genes. The investigators will study direct gene transfer by in vivo infection of rat and canine skin. They believe this research will demonstrate that gene transfer can be used for long-term treatment of this disease, and will have an application in the treatment of many other genetic disorders responding to secreted hormones and clotting factors.
The specific aims are: 1. Develop retroviral vectors to provide high-level sustained G-CSF expression in transplanted vascular smooth muscle cells and skin fibroblasts. Construct tricistronic vectors to express G-CSF, marker gene and neo to locate by histology implanted cytokine secreting cells. 2. Study porous PTFE vascular grafts seeded with transduced vascular smooth muscle cells as a means to provide therapeutic levels of G-CSF secretion in dogs. 3. Study rats and dogs to develop PTFE grafts implanted on the stomach to achieve long-term retention of vascular smooth muscle cells and expression of high-level G-CSF. 4. Investigate fibroblasts transduced with retroviral vectors employing fibronectin promoter to achieve long-term G-CSF expression when introduced as skin equivalent grafts. Study this approach in rats and dogs. 5. Study in vivo retroviral-mediated gene transfer to dog and rat skin using high-titer concentrated virus. Investigate the infected cell populations and develop this method to deliver sustained G-CSF secretion. 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 cyclic hematopoiesis and other diseases.
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