Advances in the isolation, characterization, and culture of human hematopoietic CD34+CD38- cells with extensive in vivo repopulating potential have been recently described. These developments have facilitated their evaluation as suitable targets for bone marrow transplantation and human gene therapy approaches to treat AIDS, cancer, hematological abnormalities, and inborn errors of metabolism. Unfortunately, the development of murine retrovirus-based gene transfer vectors has not kept up with these advances and progress towards clinical implementation of gene therapy protocols has lagged. Here we demonstrate that lentivirus-based vectors transduce human CD34+ and CD34+CD38- cells with high efficiency under conditions that maintain their in vivo repopulating potential. We also show that transduced CD34+CD38- cells can be induced to differentiate in culture resulting in sustained expression of the tgransgene in differentiated cell types. This grant proposal is intended to further these exciting observations in a human/mouse xenograft model. Our hypothesis is that lentivirus-based vectors are an excellent alternative for gene therapy with promise for clinical implementation. We therefore focus this proposal on the in vivo analysis of genetically modified CD34+ and CD34+CD38- cells and propose to show that after transduction these cells maintain their in vivo repopulating potential. The applicant believes that progress towards these goals will bring lentivirus-based vectors significantly closer to clinical implementation and further their utility as tools for discovery in basic research.
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