RETROVIRAL GENE TRANSFER INTO HUMAN PROGENITORS

这项研究的目标是开发一种方法，以实现和 逆转录病毒介导的人造血细胞基因转移的研究 多能祖细胞(HPPC)。这项研究是正在进行的 利用体外逆转录病毒载体开发有效方案的努力 介导性基因导入骨髓后自体骨髓移植 移植矫正致死性造血基因 精神错乱。 骨髓移植后，一小部分细胞， 造血多能祖细胞(HPPC)，可分化为干细胞 逐渐限制分化和自我更新的潜力 最终，成熟的细胞。最近的小鼠研究表明，长时间 长期、一致地转导多种血统的造血细胞 只有当基因被转移到HPPC群体中时，系统才会发生 高效率。迄今为止对逆转录病毒介导基因的研究 转移到人类造血细胞中的基因转移仅在 成熟的、承诺的干细胞群体，并表现出低基因 传输频率。本提案中描述的研究旨在 开发一种高效逆转录病毒载体的通用方法 将基因转移到人类HPPC群体中。实现这一目标的战略 涉及：1)人重组祖细胞的纯化2) 评估人类原始细胞的体外和小鼠模型的建立 祖细胞3)制定最大限度地激活 这些细胞没有伴随着重建潜力的损失4) 灵敏、特异的检测系统的建立 逆转录病毒介导的基因转移频率进入少量纯化的， 激活的祖细胞和重现前景看好的祖细胞 纯化、激活和逆转录病毒介导的基因转移方案 在灵长类动物模型中确认在实际重组中的基因转移 祖先。

The objective of this study is to develop a methodology for effecting and assessing retroviral mediated gene transfer into human hematopoietic pluripotent progenitor cells (HPPCs). This study is part of an ongoing effort to develop an effective protocol utilizing ex-vivo retroviral vector mediated gene transfer into bone marrow followed by bone marrow autologous transplantation for the correction of lethal hematopoietic genetic disorders. Following bone marrow transplantation, a small population of cells, the hematopoietic pluripotent progenitor cells (HPPCs), give rise to stem cells with progressively restricted differentiation and self renewal potential and ultimately, mature cells. Recent murine studies indicate that long term, consistent transduction of the multiple lineages of the hematopoietic system will occur only if genes are transferred into the HPPC population at high efficiency. Studies to date investigating retroviral mediated gene transfer into human hematopoietic cells have assessed gene transfer only in mature, committed stem cell populations and have demonstrated low gene transfer frequencies. The studies described in this proposal are aimed at developing a general methodology for high efficiency retroviral mediated gene transfer into the human HPPC population. The strategy to achieve this involves: 1) purification of human reconstituting progenitor cells 2) development of in vitro and murine models for assessing human primitive progenitor cells 3) development of a protocol for maximal activation of these cells without concomitant loss of reconstituting potential 4) development of sensitive and specific assay systems for determining retroviral mediated gene transfer frequency into small numbers of purified, activated progenitor cells and 5) recapitulating promising progenitor cell purification, activation, and retroviral mediated gene transfer protocols in a primate model to confirm gene transfer in actual reconstituting progenitors.