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致力于开发有效且安全的基因转导，以及针对造血细胞（包括干细胞和祖细胞和淋巴细胞）的体内操纵策略，并使用遗传标记技术来回答有关体内造血性造血的重要问题。 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使用临床实用程序。我们发现，主动循环转导的细胞具有植入缺陷，可以通过短纤维蛋白片段的短培养物来纠正，仅具有干细胞因子。高水平使我们能够继续在大型动物模型中首次跟踪对造血的克隆贡献。我们使用了一项新技术，该技术允许同时评估对外围血液种群的多个克隆贡献。我们发现，在移植后最初的1-2个月贡献了不同的雕刻细胞，然后被一组非常稳定的80多个克隆替换，这些克隆现在有助于所有谱系，现在有助于3年以上。我们使用这种强大的方法研究了细胞因子疗法，放射疗法和化学疗法对干细胞克隆体内行为的影响。到目前为止，我们已经表明，用G-CSF或SCF进行长时间的细胞因子治疗并不能显着改变导致造血的干细胞克隆数量，也不会导致可检测的克隆疲劳或募集。相比之下，低剂量的总体辐照或用链球素治疗导致干细胞克隆显着降低，导致外周血谱系。我们还开始仔细研究单个茎和祖细胞克隆的谱系贡献，询问克隆是否同等贡献每个谱系，例如粒细胞，T细胞，B细胞，B细胞，树突状细胞和肥大细胞。鉴于在两名接受基因治疗的儿童中出现白血病，该基因治疗在法国的逆转录病毒转导的造血干细胞中严重免疫缺陷，我们在恒河猴猕猴中大规模的逆转录病毒插入部位进行了大规模测序。到目前为止，尚无动物患有白血病或任何血液学疾病。插入部位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的不同动物中多次发现了几次“热点”。我们已经发现了一种新型的氧化铁颗粒，该氧化铁颗粒被所有研究的所有主要细胞类型都非特异性和高效地吸收，并且该颗粒已被用于标记，然后通过MRI Follwing通过心脏内部或静脉内注射在心肌梗死的情况下跟踪间充质干细胞。