The ability to insert genes into hematopoietic stem cells offers the potential to correct inherited diseases of the blood. There are a number of requirements for gene replacement therapy to be successful. First, vectors must be designed so that the expression of transduced gene is appropriately regulated. Secondly, the in vitro culture of the bone marrow cells while they are being transduced must not interfere with the ability of the hematopoietic stem cell to repopulate irradiated recipients and to give rise to cells of all hematopoietic lineages. We have used the mouse model to systematically improve the frequency of gene transfer to the hematopoietic stem cells. We have previously shown that the inclusion of hematopoietic growth factors in the medium used for the in vitro culture of bone marrow cells from 5-FU treated mice is required for achieving gene transfer to approximately 25% of the stem cells. Treatment of splenectomized donor animals with the combination of SCF and G-CSF in vivo results in a 40 fold increase in the number of stem cells in the peripheral blood. These stem cells, which can be collected easily and repeatedly throughout the life of the donor are more easily transduced with retroviruses than 5-FU treated bone marrow cells. To better understand the molecular biology of the hematopoietic stem cell, we have purified these cells from bone marrow by a combination of elutriation followed by removal of cells expressing lineage markers, followed by selection for high levels of c-kit expression. As few as 100 cells from these highly enriched stem cell populations are capable of repopulating recipient mice. Using RT-PCR we have examined the RNA expression of hematopoietic growth factor genes in these cell populations, in the hopes of determining an optimal combination of factors to induce division in vitro. It is hoped that by using highly purified stem cells from cytokine treated animals, gene transfer to 100% of stem cells can be achieved.
