RETROVIRAL GENE TRANSFER INTO HUMAN PROGENITORS

本研究的目的是开发一种方法， 评估逆转录病毒介导的基因转移到人造血系统中 多能祖细胞（HPPC）。这项研究是一项正在进行的 努力开发利用离体逆转录病毒载体的有效方案 介导的基因转移到骨髓中，然后骨髓自体移植 造血干细胞移植 紊乱 骨髓移植后，一小群细胞， 造血多能祖细胞（HPPC）产生干细胞 具有逐渐受限的分化和自我更新潜力 最后是成熟细胞最近的鼠类研究表明， 术语，造血干细胞的多个谱系的一致转导， 只有当基因转移到HPPC群体中时， 高效的迄今为止研究逆转录病毒介导的基因 转移到人类造血细胞中的基因转移仅在 成熟的定向干细胞群，并已证明低基因表达， 传输频率本提案中所述的研究旨在 开发高效逆转录病毒介导的通用方法 将基因转移到人HPPC群体中。实现这一目标的战略 包括：1）人重建祖细胞的纯化; 2） 用于评估人原始细胞的体外和小鼠模型的开发 祖细胞3）开发用于最大激活祖细胞的方案， 这些细胞没有伴随的重建潜力的丧失4） 开发灵敏和特异的测定系统， 逆转录病毒介导的基因转移频率， 激活的祖细胞和5）重演有希望的祖细胞 纯化、活化和逆转录病毒介导的基因转移方案 在灵长类动物模型中，以确认在实际重建中的基因转移 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.