GENE TRANSFER AND EX VIVO MANIPULATION OF HEMATOPOIETIC CELLS

造血细胞的基因转移和离体操作

• 批准号: 6290425
• 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/6290425
• 关键词: Gaucher's disease; Retroviridae; autologous transplantation; bone marrow transplantation; breast neoplasms; clinical research; clinical trials; colony stimulating factor; disease /disorder model; gene therapy; genetic markers; genetic transduction; green fluorescent proteins; hematopoiesis; hematopoietic growth factor; hematopoietic stem cells; hematopoietic tissue transplantation; human subject; human therapy evaluation; neoplasm /cancer therapy; transfection /expression vector

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. During the past year we completed an ongoing clinical trial of retroviral gene transfer into CD34+ cells from patients with breast cancer, comparing survival of cells containing a drug-resistance gene to those receiving a control gene. Cells containing the drug resistance gene survived preferentially after chemotherapy. 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 been encouraged to discover that the inclusion of the cytokine flt 3 ligand and either autologous stromal cells or fibronectin improves gene transfer efficiency into engrafting cells to levels of 10-50%, a range with clinical utility. These high levels have also allowed for the first time in a large animal model assessment of contribution of specific marked clones to different lineages over time, using inverse PCR and insertion site analysis. Over 40 clones contribute to multiple lineages for a year thus far, which has clinical implications and is a surprise based on rodent data. Other ongoing projects in the lab involve the assessment of the effect of foreign expressed genes in vector constructs on levels of gene-modified cells in vivo, using either CD34+ primitive cells or lymphocytes as targets. We have shown that tolerance to foreign gene products can be induced by introducing the gene into stem cells, as opposed to mature lymphocytes. - STEM CELLS, GENE THERAPY, RETROVIRUSES, CYTOKINES

临床和基础实验室研究旨在为造血细胞（包括干细胞和祖细胞以及淋巴细胞）开发有效和安全的基因转导和离体操作策略。在过去的一年中，我们完成了一项正在进行的临床试验，将逆转录病毒基因转移到乳腺癌患者的CD 34+细胞中，比较了含有耐药基因的细胞与接受对照基因的细胞的存活率。含有耐药基因的细胞在化疗后优先存活。在恒河猴模型中，被证明是人类临床结果的唯一预测分析，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。我们发现，加入细胞因子flt 3配体和自体基质细胞或纤连蛋白可将基因转移到移植细胞中的效率提高到10- 50%，这是一个具有临床实用性的范围。这些高水平也首次允许在大型动物模型中使用反向PCR和插入位点分析评估特定标记克隆随时间对不同谱系的贡献。到目前为止，超过40个克隆在一年内为多个谱系做出了贡献，这具有临床意义，并且是基于啮齿动物数据的惊喜。实验室正在进行的其他项目包括评估载体构建体中外源表达基因对体内基因修饰细胞水平的影响，使用CD 34+原始细胞或淋巴细胞作为靶点。我们已经证明，与成熟淋巴细胞相反，通过将基因导入干细胞可以诱导对外源基因产物的耐受性。- 干细胞，基因治疗，逆转录病毒，细胞因子