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I am part-time English faculty at a wealthy, 95 percent Anglo, liberal arts college in Pennsylvania, and I am a candidate for a PhD in Native American studies. College administrators [...]

In previous studies we showed that 5 days of treatment with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) mobilized murine repopulating cells into the peripheral blood (PB) and that these cells could be efficiently transduced with retroviral vectors. We also found that, 7-14 days after cytokine treatment, the repopulating ability of murine bone marrow (BM) increased 10-fold. In this study we examined the efficiency of gene transfer into cytokine-primed murine BM cells and extended our observations to a nonhuman primate autologous transplantation model. G-CSF/SCF-primed murine BM cells collected 7-14 days after cytokine treatment were equivalent to post-5-fluorouracil BM or G-CSF/SCF-mobilized PB cells as targets for retroviral gene transfer. In nonhuman primates, CD34-enriched PB cells collected after 5 days of G-CSF/SCF treatment and CD34-enriched BM cells collected 14 days later were superior targets for retroviral gene transfer. When a clinically approved supernatant infection protocol with low-titer vector preparations was used, monkeys had up to 5% of circulating cells containing the vector for up to a year after transplantation. This relatively high level of gene transfer was confirmed by Southern blot analysis. Engraftment after transplantation using primed BM cells was more rapid than that using steady-state bone marrow, and the fraction of BM cells having the most primitive [CD34.sup.+]/[CD38.sup.-] or [CD34.sup.+]/[CD38.sup.dim] phenotype increased 3-fold. We conclude that cytokine priming with G-CSF/SCF may allow collection of increased numbers of primitive cells from both the PB and BM that have improved susceptibility to retroviral transduction, with many potential applications in hematopoietic stem cell-directed gene therapy.

Analysis of the efficacy of recombinant adeno-associated virus (rAAV) for gene therapy using a rAAV vector with human gamma-globin gene reveals that rAAV regulates human gamma-globin expression and is effective in expressing and transferring the globin gene in primary erythroid cells for gene therapy. The genetic transfer and expression result in a high level increase of hemoglobin factor (HbF) level. Reverse transcriptase PCR assay shows that the human erythroid burst-forming unit-derived colonies contain rAAV-transduced, mutationally marked Agamma-globin gene expression, confirming its role in the gene therapy of Hb disorders, thalassemia and sickle-cell anemia.