Gene Transfer And Ex Vivo Manipulation Of Hematopoietic

基因转移和造血的离体操作

• 批准号: 6809652
• 负责人: CYNTHIA E DUNBAR
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6809652
• 关键词: Macaca mulatta; Retroviridae; blood disorder chemotherapy; cell population study; gene therapy; genetic markers; hematopoiesis; hematopoietic stem cells; hematopoietic tissue transplantation; human tissue; immunotherapy; myocardium; nonhuman therapy evaluation; radiation therapy; regeneration; stem cell transplantation; tissue /cell culture

Clinical and basic laboratory studies are directed at developing efficient and safe gene transduction and ex vivo manipulation strategies for hematopoietic cells, including stem and progenitor cells and lymphocytes, and using genetic marking techniques to answer important questions about in vivo hematopoiesis. In the rhesus model, shown to be the only predictive assay for human clinical results, we have focused on optimizing gene transfer to primitive stem and progenitor cells, and using genetic marking techniques to understand stem cell behavior in vivo.We have continued to further enhance gene transfer efficiency into rhesus engrafting cells, resulting in early levels of marked cells as high as 50-80%, with stable levels of 5-35% in all lineages, a range with clinical utility. We have found that actively-cycling transduced cells have an engraftment defect that can be corrected by a short culture on a fibronectin fragment with stem cell factor alone. The high levels have allowed us to continue to track the clonal contributions to hematopoiesis for the first time in a large animal model. We have utilized a new technology that allows simultaneous assessment of multiple clonal contributions to peripheral blood populations. We have found a different population of engrafting cells that contribute for the first 1-2 months post-transplantation, that are then replaced by a very stable set of over 80 clones that contribute to all lineages now for over 3 years. We have investigated the impact of cytokine therapy, radiation, and chemotherapy on the in vivo behavior of stem cell clones, using this powerful methodology. Thus far we have shown that prolonged cytokine treatment with either G-CSF or SCF does not significantly alter the number of stem cell clones contributing to hematopoiesis, nor result in detectable clonal exhaustion or recruitment. In contrast, treatment with low dose total body irradiation or with busulfan results in a significant decrease in stem cell clones contributing to peripheral blood lineages. We have also begun to carefully investigate the lineage contributions of individual stem and progenitor cell clones, asking whether clones contribute equally to each lineage such as granulocytes, T cells, B cells, dendritic cells and mast cells. Given the occurence of leukemia in two children receiving gene therapy for severe immunodeficiencies with retrovirally-transduced hematopoietic stem cells in France, we are engaged in large scale sequencing of retroviral insertion sites in rhesus macaques transplanted with transduced cells. Thus far, no animal has developed leukemia or any hematologic disorder. The insertion site analysis shows non-random preference for insertions within genes, and several "hot spots" found multiple times in different animals within transcription factors previously implicated in leukemias. We have discovered a novel iron oxide particle that is taken up nonspecifically and highly efficiently by all primary cell types studied, and this particle has been utilized to label and then track mesenchymal stem cells in vivo via MRI follwing intracardiac or intravenous injection in the setting of a myocardial infarction.

临床和基础实验室研究旨在开发高效、安全的造血细胞（包括干细胞、祖细胞和淋巴细胞）基因转导和体外操作策略，并使用遗传标记技术来回答有关体内造血的重要问题。在恒河猴模型中，被证明是人类临床结果的唯一预测试验，我们专注于优化基因转移到原始干细胞和祖细胞，并使用遗传标记技术来了解干细胞在体内的行为。我们继续进一步提高基因转移到恒河移植细胞中的效率，导致标记细胞的早期水平高达50-80%，所有谱系的稳定水平为5-35%，这一范围具有临床应用价值。我们发现，主动循环转导的细胞有一种移植缺陷，这种缺陷可以通过单独使用干细胞因子在纤维连接蛋白片段上进行短时间培养来纠正。高水平使我们能够第一次在大型动物模型中继续追踪克隆对造血的贡献。我们利用了一种新技术，可以同时评估多克隆对外周血群的贡献。我们发现移植细胞的不同群体在移植后的最初1-2个月起作用，然后被一组非常稳定的80多个克隆所取代，这些克隆对所有谱系都有贡献，现在超过3年。我们已经研究了细胞因子治疗，放疗和化疗对干细胞克隆体内行为的影响，使用这种强大的方法。到目前为止，我们已经证明，使用G-CSF或SCF进行长时间的细胞因子治疗不会显著改变造血干细胞克隆的数量，也不会导致可检测到的克隆耗尽或募集。相比之下，低剂量全身照射或布硫丹治疗可显著减少有助于外周血系的干细胞克隆。我们也开始仔细研究单个干细胞和祖细胞克隆的谱系贡献，询问克隆对每个谱系的贡献是否相等，如粒细胞、T细胞、B细胞、树突状细胞和肥大细胞。鉴于法国两名儿童因严重免疫缺陷而接受逆转录病毒转导造血干细胞基因治疗后发生白血病，我们正在对移植了转导细胞的恒河猴进行逆转录病毒插入位点的大规模测序。到目前为止，还没有动物患上白血病或任何血液病。插入位点分析显示了基因内插入的非随机偏好，并且在先前与白血病有关的转录因子中多次在不同动物中发现了几个“热点”。我们已经发现了一种新的氧化铁颗粒，它被所有研究的原代细胞类型非特异性地高效地吸收，并且该颗粒已被用于在心肌梗死的情况下，通过心内或静脉注射后的MRI标记和跟踪体内间充质干细胞。