GENE THERAPY OF BLOOD DISEASES

血液病的基因治疗

• 批准号: 2225643
• 负责人: Clinton D Lothrop
• 金额: $ 23.48万
• 依托单位: AUBURN UNIVERSITY AT AUBURN
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-10 至 1997-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2225643
• 关键词: blood disorder; bone marrow; cell cycle; colony stimulating factor; complementary DNA; disease /disorder model; dogs; fluorouracil; gene therapy; genetic transduction; hematopoietic growth factor; hematopoietic stem cells; hemolytic anemia; interleukin 1; interleukin 3; interleukin 6; mixed tissue /cell culture; molecular cloning; mutant; nucleic acid sequence; polymerase chain reaction; pyruvate kinase deficiency; transfection /expression vector

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.

该项目的长期目标是调查潜力 逆转录病毒载体介导基因的应用及安全性测定 使用犬溶血模型转移治疗血液疾病 丙酮酸激酶（PK）缺乏引起的贫血。 第一个主要目标 该项目旨在开发逆转录病毒转导/骨髓培养 造血干中外源基因高效表达的方案 细胞。 表达人类多药耐药性（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 细胞将与造血生长因子（IL-1、IL-3、IL- 6、IL-11、G-CSF、干细胞因子）激活造血干细胞 从 进入细胞周期。 然后使用 co-转导细胞 培养、上清液和长期骨髓培养物转导 协议。 在另外的实验中，动物将接受 5- 获得前进行氟尿嘧啶 (250 mg/m2) 或亚致死照射 (2.5 Gy) 骨髓用于逆转录病毒转导以激活造血干 细胞。 逆转录病毒转导的效率将使用以下方法确定 体外集落形成单位测定和聚合酶链反应 DNA 分析。 骨髓重建和MDR基因的表达 体内也将用作确定最有效的终点 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测定。 第二专业 目标是使用含有犬 R 型 PK cDNA 的逆转录病毒载体 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、红细胞形态、网织红细胞计数、红细胞 t1/2） PK 酶水平将用于评估接受治疗的 PK 缺陷犬 与基因疗法。 个别动物将受到密切监测 自发性疾病并定期检查辅助病毒以评估 基因疗法治疗血液疾病的安全性。 独特的 该提案的优点是其他地方没有的能力 研究大型动物红色基因治疗的有效性和安全性 使用骨髓培养/逆转录病毒转导的血细胞疾病模型 与预期在人体临床试验中使用的方案类似。 拟议的研究将提供有关以下方面的重要新信息： 基因疗法在无需人类治疗的血液疾病中的应用 风险或痛苦。