The goal of this project is to find effective vectors for delivery and expression of foreign genes in hematopoietic stem cells and their differentiated progeny cells. A retroviral vector (pSFF) derived from murine Friend spleen focus forming virus was used to transduce murine hematopoietic stem cells and express a cell surface marker protein, mutated murine prion protein, in vitro and in vivo after transplantation. To enhance retroviral vector integration in bone marrow cells, mice were treated with 5-fluorouracil (5-FU) to increase stem cell mitotic activity which peaked on day 8 post-5-FU. The infectivity titer of the vector, pSFF-mPrP-3F4, was determined by a novel assay in which antigen-positive foci of infected cells were detected after replication and spread of the vector in cultures of mixed packaging cell lines. Infection of Sca-1+/Lineageneg- low cells with pSFF-mPrP-3F4 resulted in marker protein expression in 40% of the progeny cells after 7 days of culture. Transplantation of marrow cells or sorted Sca-1+/Lineage neg-low cells transduced with vector resulted in 3F4-positive mPrP expression in 11-37% of donor-derived peripheral blood leukocytes at two weeks. Though the percentage of 3F4- positive blood cells gradually declined, at 28 weeks 23% of recipient mice still maintained expression of the marker gene. Expression was observed in lymphoid, myeloid and erythroid lineages and was detected in Sca-1+/Lineage neg-low marrow cells. The multi-lineage, high frequency expression observed suggests that pSFF may have utility in gene therapy directed to hematopoietic stem cells and their differentiated progeny.
