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.
Showing the most recent 10 out of 13 publications
