GENE THERAPY OF BLOOD DISEASES

这个项目的长期目标是调查潜在的 逆转录病毒载体介导基因的应用及安全性检测 利用犬溶血模型移植治疗血液病 由于丙酮酸激酶(PK)缺乏而引起的贫血。第一个主要目标 这个项目是为了发展逆转录病毒转导/骨髓培养 外源基因在造血干细胞中的高效表达方案 细胞。一种表达人多药耐药基因的逆转录病毒载体 基因将被用于这些研究。为了实现这一目标，骨髓 细胞将与造血生长因子(IL-1、IL-3、IL-3)共同孵育。 6、IL-11、G-CSF、干细胞因子)激活造血干细胞 进入细胞周期。然后，细胞将被转换为使用co- 培养、培养上清液和长期骨髓培养转导 协议。在更多的实验中，动物将被5- 氟尿嘧啶(250 mg/m2)或亚致死剂量照射(2.5Gy. 用于逆转录病毒转导激活造血干细胞的骨髓 手机。逆转录病毒转导的效率将通过 体外集落形成单位试验和聚合酶链式反应 DNA分析。骨髓重建及多药耐药基因的表达 体内也将被用作终点，以确定最有效的 逆转录病毒转导方案，因为这是目前唯一 犬造血干细胞的可靠检测方法。第二大调 目的是利用含犬R型PK基因的逆转录病毒载体 最有效的逆转录病毒转导方案在 研究PK缺乏症基因治疗的初步实验。标准 血液学参数(CBC、RBC形态、网织红细胞计数、RBC T1/2) PK酶水平将被用来评估治疗后的PK缺陷犬 用基因疗法。每只动物都将受到严密监测 自发性疾病并定期检查辅助病毒以进行评估 基因治疗在血液疾病治疗中的安全性。独一无二的 这项提议的优点是能够 研究基因治疗在大型动物红斑狼疮中的有效性和安全性 应用骨髓培养/逆转录病毒转导技术建立血细胞病模型 类似于预期将用于人体临床试验的方案。 拟议的研究将提供有关 基因治疗在非人类血液病治疗中的适用性 风险或痛苦。

The long range goal of this project is to investigate the potential application and determine the safety of retroviral vector mediated gene transfer for treatment of blood diseases using a canine model of hemolytic anemia due to pyruvate kinase (PK) deficiency. The first major goal of this project is to develop a retroviral transduction/bone marrow culture protocol for efficient expression of exogenous genes in hematopoietic stem cells. A retroviral vector expressing the human multidrug resistance (MDR) gene will be used in these studies. To accomplish this goal bone marrow cells will be incubated with hematopoietic growth factors (IL-1, IL-3, IL- 6, IL-11, G-CSF, stem cell factor) to activate the hematopoietic stem cell from Go into the cell cycle. Cells will then be transduced using co- cultivation, supernatant and long-term bone marrow culture transduction protocols. In additional experiments animals will be treated with 5- fluorouracil (250 mg/m2) or sublethal irradiation (2.5 Gy) before obtaining bone marrow for retroviral transduction to activate the hematopoietic stem cell. The efficiency of retroviral transduction will be determined using an in vitro colony forming unit assay and by polymerase chain reaction analysis of DNA. Bone marrow reconstitution and expression of the MDR gene in vivo will also be used as the endpoint to determine the most efficient retroviral transduction protocol because that is presently the only reliable assay for the canine hematopoietic stem cell. The second major goal is to use a retroviral vector containing the canine R-type PK cDNA in the most efficient retroviral transduction protocol determined in the initial experiments to investigate gene therapy of PK deficiency. Standard hematologic parameters (CBC, RBC morphology, reticulocyte count, RBC t1/2) and PK enzyme levels will be used to evaluate PK deficient dogs treated with gene therapy. Individual animals will be closely monitored for spontaneous disease and periodically checked for helper virus to evaluate the safety of gene therapy in the treatment of blood diseases. The unique strengths of this proposal, not available elsewhere are the ability to investigate the efficacy and safety of gene therapy in a large animal red blood cell disease model using bone marrow culture/retroviral transduction protocols similar to that expected to be used in human clinical trials. The proposed studies will provide important new information on the applicability of gene therapy to treatment of blood diseases without human risk or suffering.