GENE TRANSFER AND EX VIVO MANIPULATION OF HEMATOPOIETIC CELLS

临床和基础实验室研究旨在开发有效和安全的造血细胞基因转导和体外操作策略，包括干细胞和祖细胞和淋巴细胞。在过去的一年里，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CD34+细胞中，比较含有耐药基因的细胞和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。我们鼓舞地发现，细胞因子flt 3配体与自体基质细胞或纤维连接蛋白的结合可将移植细胞的基因转移效率提高到10-50%，这一范围具有临床应用价值。这些高水平也首次允许在大型动物模型中评估特定标记克隆随着时间推移对不同谱系的贡献，使用反向PCR和插入位点分析。到目前为止，超过40个克隆在一年内贡献了多个谱系，这具有临床意义，并且基于啮齿动物数据是一个惊喜。实验室中其他正在进行的项目包括评估载体结构中外源表达基因对体内基因修饰细胞水平的影响，使用CD34+原始细胞或淋巴细胞作为靶点。我们已经证明，对外来基因产物的耐受性可以通过将基因引入干细胞而不是成熟淋巴细胞来诱导。-干细胞，基因疗法，逆转录病毒，细胞因子

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