CANINE G-CSF GENE TRANSFER

这项研究的目的是证明 逆转录病毒载体基因转移治疗灰牧羊犬的实验研究 循环造血法。周期性造血是一种罕见的疾病， 在人类和灰色牧羊犬中均可发生，其特点是周期性 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。反复出现的严重中性粒细胞减少症导致 细菌感染和寿命缩短。循环 造血可以通过骨髓治愈或转移 移植，表明干细胞缺陷是移植的来源 疾病。在受影响的狗和人身上，长期服用 重组G-CSF产生持续性中性粒细胞增多症并消除 反复的造血循环。我们建议使用编码G-的矢量 脑脊液感染自体靶细胞作为基因转移的载体。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，选择容易获取，培养， 可移植性和允许稳定表达的潜力 转导基因。我们将通过体内研究直接基因转移 犬角质形成细胞感染。这些研究采用了适当的 一种人类造血疾病的犬模型。总体目标 这一应用的目的是证明基因转移可以用于 这种遗传病和其他遗传病的长期治疗。具体的 目的是：(1)构建含犬的可选择逆转录病毒载体 多种增强子和启动子调控下的G-CSFc DNA。(2A) 感染培养的血管平滑肌细胞，检测G-CSF的表达。 将受感染的/选定的自体细胞移植到狗的颈动脉和 监测静脉血G-CSF的持续时间和表达情况。活体监护仪 CBC转导基因表达、细胞因子水平与健康的关系 关于动物的。(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 感染狗的自体皮肤成纤维细胞并监测其 粒细胞集落刺激因子的持续和表达。(2C)感染犬骨髓 细胞体外培养及G-CSF在造血细胞中表达的检测 祖细胞。将受感染的自体骨髓移植到狗体内 监测造血祖细胞G-CSF的表达，以及 外周血细胞的产生。(3)犬的体内感染 角质形成细胞使用浓缩纯化的高滴度病毒。

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.