Gene replacement therapy has several potential therapeutic applications. Addition of a structurally normal human beta globin gene into bone marrow cells could correct the deficiency of beta globin synthesis in patients with beta thalassemia or replace the defective beta globin in the hemoglobin of patients with sickle cell disease. Retrovirus mediated gene transfer is the most efficient means to introduce new genetic material into eukaryotic cells. We have previously shown that a human beta globin gene transferred via a retrovirus into mouse bone marrow stem cells is expressed in mature erythroid cells, and not in other myeloid or lymphoid cells. We have defined the optimal conditions for gene transfer to mouse bone marrow stem cells. These conditions include pretreatment of the donor animals with 5-fluorouracil (5-FU) and culture of the donor bone marrow cells for 6 days in the presence of interleukin-3 (IL-3) and interleukin-6. Using our extremely high titer (<10(10) retrovirus particles/ml) amphotropic retrovirus producer clone we have successfully achieved gene transfer in 7 of 7 Rhesus monkeys reconstituted with infected bone marrow cells. The provirus has persisted in 3 of 3 animals analyzed between 95 and 110 days. These results demonstrate reproducible gene transfer to extremely primitive bone marrow progenitor cells in a large animal model, an important first step towards globin gene transfer to human patients. Our current focus is to optimize conditions for gene transfer to primates based on our results in mice, and to include the appropriate regulatory sequences for high level expression of the human beta globin gene in transduced cells.
