Cytokine-mobilized peripheral blood CD34⁺Thy-1⁺ Lin^- human hematopoietic stem cells as target cells for transplantation-based gene therapy

Gene-therapy of blood-borne disorders may be best achieved using hematopoietic stem cells (HSC) which have extensive self renewal potential as well as multillneage repopulatlng potential as a cellular target. The human HSC, which is CD34⁺Thy-1⁺Lin^- has been isolated from fetal, adult bone marrow and cytokine-mobilized peripheral blood (MPB) (1-3). Results presented in this study show that the degree of mobilization of HSC into preipheral blood of cancer patients is highly variable and that the combined use of high dose chemotherapy and GM-CSF as a mobi-lization strategy is superior to the use of G-CSF with regardto the mobilization of true HSC. A multistep cell isolation procedure has been developed which utilizes high speed flow-cytometric cell sorting and allows the isolation of sufficient numbers of HSC from MPB to permit their use as an hematopoietic graft for clinical transplantation. Hematopoietic stem cells isolated from MPB are capable of selfrenewal and differentiation Into multiple hematopoietic lineages as shown by their behavior In both in vitro and in vivo assays. Mobilized PB mononuclear cells isolated from cancer patients are frequently contaminated with tumor cells. Using this cell Isolation procedure, HSC preparations from patients with multiple myeloma have been created with greatly reduced tumor cell burdens. These CD34⁺Thy-1⁺Lin^- cells are capable of being stably transduced at high efficiency (32-75%) by co-culture on a cell line producing recombinant retroviruses containing the neomycinresistant gene. These HSC cell populations are likely ideal targets for hematopoietic cell-based gene therapy. Purified CD34⁺Thy-1 ⁺ Lin^- HSC isolated from MPB, with or without gene-modification may serve as tumor-free autografts, the use of which may prolong the disease-free and overall survival of patients with various malignancies undergoing autotransplantation.