CANINE G-CSF GENE TRANSFER

描述：本研究的目的是证明 逆转录病毒介导的基因转移作为治疗灰色牧羊犬狗， 周期性造血 重组犬G-CSF的长期给药 消除了患病狗的反复造血循环。 奥斯本博士 和他的同事们建议使用编码犬G-CSF cDNA的载体， 将自体靶细胞作为基因转移的载体， 在体内感染靶细胞。 他们将研究植入血管 人造PTFE移植物和PTFE胃植入物中的平滑肌细胞， 提供G-CSF的安全长期表达。 皮肤成纤维细胞将 使用大鼠纤维连接蛋白的成分作为皮肤等效移植物移植 启动子表达G-CSF并克服逆转录病毒载体失活。 选择这些靶细胞是为了便于接近、培养， 可移植性和允许稳定表达转导的 基因. 研究人员将研究直接基因转移， 大鼠和犬皮肤感染。 他们相信这项研究将 证明基因转移可以用于长期治疗这种疾病， 疾病，并将在许多其他遗传性疾病的治疗中应用。 对分泌的激素和凝血因子有反应的疾病。 的 具体目标是：1. 开发逆转录病毒载体， 在移植的血管平滑肌细胞中持续表达G-CSF， 皮肤成纤维细胞 构建三顺反子载体表达G-CSF、标志物 通过组织学方法对植入的细胞因子分泌细胞进行基因和neo定位。 2. 多孔聚四氟乙烯血管移植物与转导血管平滑肌的研究 肌肉细胞作为提供治疗水平的G-CSF分泌的手段， 狗 3. 研究大鼠和狗的PTFE移植物植入 胃，以实现长期保留血管平滑肌细胞和 高水平G-CSF的表达。 4. 研究转导有 使用纤连蛋白启动子获得长期G-CSF的逆转录病毒载体 当作为皮肤等效移植物引入时表达。 研究这种方法 在老鼠和狗身上。 5. 研究体内逆转录病毒介导的基因转移 使用高滴度浓缩病毒的狗和大鼠皮肤。 探讨 感染的细胞群，并开发这种方法来提供持续的G-CSF 分泌物 这些研究采用适当的犬模型， 造血系统疾病 本申请的总体目标是 证明基因转移可用于长期治疗周期性 造血和其他疾病。

DESCRIPTION: 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.