CANINE G-CSF GENE TRANSFER

本研究的目的是证明 逆转录病毒介导的灰牧羊犬基因转移治疗 周期性造血 循环性造血是一种罕见的疾病， 人和灰色牧羊犬犬都有发生， 血液中性粒细胞，单核细胞，淋巴细胞，嗜酸性粒细胞， 网织红细胞和血小板 复发性严重中性粒细胞减少症导致 细菌感染和预期寿命缩短。 环状 造血可以通过骨髓治愈或转移 移植，表明干细胞缺陷作为起源， 疾病 在受影响的狗和人中， 重组G-CSF产生持续的嗜中性粒细胞，并消除 复发性造血循环 我们建议使用矢量编码G- CSF感染自体靶细胞作为载体进行基因转移。 我们 将研究三种细胞类型，血管平滑肌细胞，皮肤 成纤维细胞和骨髓细胞，选择这些细胞是为了易于获取，培养， 可移植性和允许允许稳定表达 转导基因。 我们将研究直接基因转移，通过在体内 犬角质细胞感染。 这些研究采用适当的 人类造血系统紊乱的犬模型。 总体目标 本申请的目的是证明基因转移可用于 长期治疗这种疾病和其他遗传疾病。 具体 目的：（1）构建含犬源基因的选择性逆转录病毒载体 G-CSF cDNA在各种增强子和启动子的控制下。（2a） 感染培养的血管平滑肌细胞并测定G-CSF表达。 将感染/选择的自体细胞移植到狗颈动脉中， 并监测G-CSF的持续性和表达。 监测体内 通过CBC、细胞因子水平和健康对转导基因表达的影响 动物。(2b)用这些载体感染犬皮肤成纤维细胞， 测定G-CSF的表达和分泌水平。 移植 感染自体皮肤成纤维细胞到狗，并监测其 G-CSF的持续存在和表达。(2c)感染犬骨髓 体外细胞培养和造血干细胞中G-CSF表达试验 祖细胞 将感染的自体骨髓移植到狗体内， 监测造血祖细胞的G-CSF表达，和 外周血细胞的产生。(3)犬体内感染 使用浓缩纯化的高滴度病毒的角质形成细胞。

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.