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、安全的造血细胞基因转导和体外操作策略，包括干细胞、祖细胞和淋巴细胞，并使用基因标记技术来回答体内造血的重要问题。在恒河猴模型中，我们专注于优化向原始干细胞和祖细胞的基因转移，并使用遗传标记技术来了解干细胞在活体中的行为。我们继续提高向恒河猴移植细胞的基因转移效率，导致标记细胞的早期水平高达50%-80%，在所有谱系中稳定在5%-35%的水平，这一范围具有临床实用价值。我们发现，主动循环的转导细胞有一个植入缺陷，可以通过在纤维连接蛋白片段上单独使用干细胞因子进行短期培养来纠正。高水平使我们第一次能够在大型动物模型中继续跟踪克隆对造血的贡献。我们利用了一种新技术，可以同时评估对外周血群体的多个克隆性贡献。我们发现了一个不同的移植细胞群体，它们在移植后的1-2个月内做出贡献，然后被一组非常稳定的80多个克隆所取代，这些克隆现在已经为所有血统贡献了3年以上。我们使用这一强大的方法研究了细胞因子治疗、放射治疗和化疗对干细胞克隆体内行为的影响。到目前为止，我们已经证明，长期使用G-CSF或SCF的细胞因子治疗不会显著改变有助于造血的干细胞克隆的数量，也不会导致可检测到的克隆耗尽或重新募集。相比之下，低剂量全身照射治疗导致外周血系干细胞克隆显著减少。有关丁硫丹的研究正在进行中。我们还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包括内皮和肌肉在内的其他组织的贡献，并建立了恒河猴心肌梗死模型，该模型允许我们评估在梗死周通过细胞因子动员原始细胞是否能够改善心肌梗死后的心肌再生和功能。到目前为止，没有证据表明有积极的好处，但我们继续研究不同的动员剂和动员计划。我们发现了一种新的氧化铁颗粒，它可以被所研究的所有原代细胞高效地非特异性地摄取，并且这种颗粒已经被用来标记并跟踪体内的间充质干细胞，在心肌梗死时，可以通过心脏内或静脉注射进行核磁共振成像。