GENE THERAPY OF BLOOD DISEASES

该项目的长期目标是研究潜在的 逆转录病毒载体介导的基因的应用和安全性测定 使用犬溶血模型转移用于治疗血液病 丙酮酸激酶（PK）缺乏引起的贫血。 第一个主要目标 本项目是开发一种逆转录病毒转导/骨髓培养 在造血干细胞中有效表达外源基因的方案 细胞 表达人多药耐药（MDR）的逆转录病毒载体 基因将用于这些研究。 为了实现这一目标，骨髓 将细胞与造血生长因子（IL-1，IL-3，IL-10，IL-11，IL-12，IL-13，IL-14，IL-15，IL-16，IL-17，IL-18，IL-19， 6、IL-11、G-CSF、干细胞因子）以激活造血干细胞 进入细胞周期。 然后将细胞用共转染。 培养、上清液和长期骨髓培养转导 协议. 在另外的实验中，动物将用5- 氟尿嘧啶（250 mg/m2）或亚致死剂量照射（2.5戈伊） 骨髓用于逆转录病毒转导以激活造血干细胞 cell. 逆转录病毒转导的效率将使用以下测定： 体外集落形成单位测定和聚合酶链反应 DNA分析。 骨髓重建与MDR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，因为这是目前唯一的 犬造血干细胞的可靠检测。 第二主 目的是使用含有犬R型PK cDNA的逆转录病毒载体， 中确定的最有效的逆转录病毒转导方案 研究PK缺陷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） 和PK酶水平将用于评价PK缺陷型治疗犬 基因治疗 将密切监测个体动物的 自发性疾病，并定期检查辅助病毒， 基因治疗血液病的安全性。 独特的 这一建议的优势，没有其他地方提供的能力， 探讨基因治疗对大型动物红细胞的有效性和安全性 使用骨髓培养/逆转录病毒转导的血细胞疾病模型 类似于预期用于人类临床试验的方案。 拟议中的研究将提供关于 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.