CANINE G-CSF GENE TRANSFER

描述：这项研究的目标是证明其有效性 逆转录病毒介导的基因转移治疗灰牧羊犬的研究 循环造血法。犬重组粒细胞集落刺激因子长期应用的研究 取消受影响的狗反复的造血循环。奥斯本博士 和他的同事们提议使用编码犬G-CSF cDNA的载体来 转导自体靶细胞作为基因转移的载体和 体内感染靶细胞。他们将研究血管植入 聚四氟乙烯假体和聚四氟乙烯胃植入物中的平滑肌细胞 提供长期安全的G-CSF表达。皮肤成纤维细胞将是 使用大鼠纤维连接蛋白成分作为皮肤等值移植物进行移植 启动子表达G-CSF，克服逆转录病毒载体失活。 选择这些靶细胞是为了便于获取、培养 可移植性和允许转导基因稳定表达的潜力 基因。研究人员将通过体内研究直接基因转移。 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这种疾病的长期治疗 疾病，并将在治疗许多其他基因 对分泌的荷尔蒙和凝血因子有反应的紊乱。这个 具体目标是：1.开发逆转录病毒载体，提供高水平的 G-CSF在移植血管平滑肌细胞中的持续表达 皮肤成纤维细胞。构建三顺反子载体表达G-CSF标记物 基因和neo通过组织学定位植入细胞因子分泌细胞。2. 转导血管光滑的多孔PTFE血管移植物的研究 肌肉细胞作为一种手段提供治疗水平的G-CSF在 狗。3.研究大鼠和犬移植聚四氟乙烯的方法 实现胃部血管平滑肌细胞的长期留存 高水平G-CSF的表达。4.研究转导成纤维细胞 利用纤维连接蛋白启动子获得长期G-CSF的逆转录病毒载体 当被介绍为皮肤等效移植物时的表情。研究这一方法 在老鼠和狗身上。5.体内逆转录病毒介导的基因转移研究 狗和鼠皮肤使用高滴度浓缩病毒。调查 感染的细胞群并开发这种方法来传递持续的G-CSF 分泌物。这些研究使用了一个合适的人类犬类模型 血液病。此应用程序的总体目标是 证明基因转移可用于周期性疾病的长期治疗 造血等疾病。

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.