TRANSDUCTION OF BABOON HEMATOPOIETIC STEM CELLS

狒狒造血干细胞的转导

• 批准号: 6202423
• 负责人: ROBERT GOFF ANDREWS
• 金额: $ 20.18万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6202423
• 关键词: CD34 molecule; Retroviridae; SDS polyacrylamide gel electrophoresis; adeno associated virus group; athymic mouse; baboons; bone marrow; cell differentiation; cell growth regulation; colony stimulating factor; cytokine; flow cytometry; fluorescent dye /probe; fluorouracil; gene expression; gene therapy; genetic transduction; hematopoiesis; hematopoietic growth factor; hematopoietic stem cells; high performance liquid chromatography; laboratory mouse; monoclonal antibody; polymerase chain reaction; tissue /cell culture; transfection /expression vector

The effective application of gene therapy to human hematopoietic diseases continues to be hampered by the inability to introduce genetic material into a sufficiently large proportion of hematopoietic stem cells. Efficient transduction of murine hematopoietic stem cells has been achieved by treating mice with 5-fluorouracil (5-FU) or with the combination of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) to elicit stem cell proliferation before collecting cells. In large animal models and humans it is has not yet been possible to transduce a sufficiently high proportion of marrow repopulating stem cells to potentially effect a therapeutic benefit. We hypothesize that, as in mice, the induction of proliferation of hematopoietic stem cells in humans will be required for effective gene transduction by retroviral vectors. Using baboons as a model, we have demonstrated that lymphohematopoietic stem cells express the CD34 antigen, that stem cells are mobilized into the peripheral blood by treatment with SCF or with SCF plus G-CSF, and that the transduction efficiency of marrow CD34+ progenitors by retroviral vectors is increased 10-fold 6 days after 5-FU treatment. We hypothesize that perturbing hematopoiesis in baboons with cytokine combinations and/or cytotoxic drugs will elicit CD34+ stem cell populations more susceptible to transduction by retroviral and possibly AAV vectors. Therefore, the SPECIFIC AIMS of this project are to 1) determine if treatment of baboons with SCF and G-CSF alone or in combination, or 5-FU alone or in combination with SCF and G-CSF will increase the ability to transduce CD34+ stem cells from bone marrow and peripheral blood using murine amphotropic retroviral vectors, and to compare these results with those obtained with CD34+ cord blood cells. 2) Determine if CD34+ stem cells from marrow, mobilized blood, and cord blood are more susceptible to transduction by gibbon ape leukemia virus and AAV vectors as compared to murine amphotropic vectors. 3) Determine if genetically altered CD34+ cells from different sources differ in their capacity to reconstitute lymphohematopoiesis in vivo. These studies will therefore define the conditions necessary to achieve effective gene transfer into hematopoietic stem cells in baboons and will provide further insights directly applicable to gene therapy in humans.

基因治疗在人类造血系统疾病中的有效应用 继续受到无法引入遗传物质的阻碍 转化为足够大比例的造血干细胞。 小鼠造血干细胞的高效转导 通过用 5-氟尿嘧啶 (5-FU) 或用 干细胞因子 (SCF) 和粒细胞集落刺激因子的组合 因子（G-CSF）在收集细胞之前引发干细胞增殖。 在大型动物模型和人类中，目前还不可能 转导足够高比例的骨髓再生干细胞 潜在地产生治疗效果。我们假设，如 小鼠，诱导人类造血干细胞增殖 通过逆转录病毒载体进行有效的基因转导需要。 以狒狒为模型，我们证明了淋巴造血功能 干细胞表达 CD34 抗原，干细胞被动员到 通过用SCF或用SCF加G-CSF处理来收集外周血，以及 逆转录病毒对骨髓 CD34+ 祖细胞的转导效率 5-FU治疗后6天载体增加10倍。我们假设 用细胞因子组合干扰狒狒的造血功能和/或 细胞毒性药物将使 CD34+ 干细胞群更容易受到影响 通过逆转录病毒和可能的 AAV 载体进行转导。因此， 该项目的具体目标是 1) 确定是否对狒狒进行治疗 单独或组合使用SCF和G-CSF，或单独或组合使用5-FU 与 SCF 和 G-CSF 组合将增加转导能力 使用小鼠骨髓和外周血提取 CD34+ 干细胞 双嗜性逆转录病毒载体，并将这些结果与那些结果进行比较 用 CD34+ 脐带血细胞获得。 2) 确定是否CD34+干细胞 骨髓、动员血和脐带血更容易受到 与长臂猿白血病病毒和 AAV 载体的转导相比 鼠兼嗜性载体。 3) 确定CD34+是否经过基因改造 不同来源的细胞重组能力不同 体内淋巴造血作用。因此，这些研究将定义 实现有效基因转移至造血系统所需的条件 狒狒干细胞并将直接提供进一步的见解 适用于人类基因治疗。