Gene Transfer And Ex Vivo Manipulation Of Hematopoietic

基因转移和造血的离体操作

• 批准号: 6690539
• 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/6690539
• 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 results in a significant decrease in stem cell clones contributing to peripheral blood lineages. Studies with busulfan are ongoing. 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. We have begun to study the contributions of these clones to other tissues, including endothelium and muscle, and have developed a rhesus macaque myocardial infarction model that has allowed us to assess whether mobilization of primitive cells via cytokines in the peri-infarct period results in improved myocardial regeneration and function post MI. Thus far, there is no evidence for a positive benefit, but we continue to study different agents and schedules for mobilization. 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，低剂量全身照射治疗可显著减少有助于外周血系的干细胞克隆。有关丁硫丹的研究正在进行中。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。我们已经开始研究这些克隆对其他组织的贡献，包括内皮和肌肉，并建立了一个恒河猴心肌梗死模型，该模型使我们能够评估梗死周围时期通过细胞因子动员原始细胞是否会改善心肌再生和心肌梗死后的功能。到目前为止，没有证据表明有积极的益处，但我们将继续研究不同的动员剂和时间表。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。