HIGH-FREQUENCY CELL-SURFACE EXPRESSION OF A FOREIGN PROTEIN IN MURINEHEMATOPOIETIC STEM-CELLS USING A NEW RETROVIRAL VECTOR

A retroviral vector (pSFF) derived from murine Friend spleen focus for ming virus was used to transduce murine hematopoietic stem cells and e xpress 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-fluorourac il (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 d etermined by a novel assay in which antigen-positive foci of infected cells were detected after replication and spread of the vector in cult ures of mixed packaging cell lines. Infection of Sca-1+/Lineage(neg-lo w) cells with pSFF-mPrP-3F4 resulted in marker protein expression in 4 0% of the progeny cells after 7 days of culture. Transplantation of ma rrow cells or sorted Sca-1(+)/Lineage(neg-low) cells transduced with v ector resulted in 3F4-positive mPrP expression in 11% to 37% of donor- derived peripheral blood leukocytes at 2 weeks. Though the percentage of 3F4-positive blood cells gradually declined, at 28 weeks 23% of rec ipient mice still maintained expression of the marker gene. Expression was observed in lymphoid, myeloid, and erythroid lineages and was det ected in Sca-1+/Lineage(neg-low) marrow cells. The multilineage, high- frequency expression observed suggests that pSFF may be useful in gene therapy directed at hematopoietic stem cells and their differentiated progeny. (C) 1996 by The American Society of Hematology.