CYTOKINE-MOBILIZED PERIPHERAL-BLOOD CD34(-1(+)LIN(-) HUMAN HEMATOPOIETIC STEM-CELLS AS TARGET-CELLS FOR TRANSPLANTATION-BASED GENE-THERAPY()THY)

Gene-therapy of blood-borne disorders may be best achieved using hemat opoietic stem cells (HSC) which have extensive self renewal potential as well as multilineage repopulating potential as a cellular target. T he human HSC, which is CD34(+)thy-1(+)Lin(-) has been isolated from fe tal, adult bone marrow and cytokine-mobilized peripheral blood (MPB) ( 1-3). Results presented in this study show that the degree of mobiliza tion of HSC into preipheral blood of cancer patients is highly variabl e and that the combined use of high dose chemotherapy and GM-CSF as a mobilization strategy Is superior to the use of G-CSF with regard to t he 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 perm it their use as an hematopoietic graft for clinical transplantation. H ematopoietic stem cells isolated from MPB are capable of self-renewal and differentiation into multiple hematopoietic lineages as shown by t heir behavior ire both in vitro and In vivo assays. Mobilized PB monon uclear cells isolated from cancer patients are frequently contaminated with tumor cells. Using this cell isolation procedure, HSC preparatio ns from patients with multiple myeloma have been created with greatly reduced tumor cell burdens. These CD34(+)Thy-1(+)Lin(-) cells are capa ble of being stably transduced at high efficiency (32-75%) by co-cultu re on a cell line producing recombinant retroviruses containing that n eomycin-resistant gene. These HSC call populations are likely ideal ta rgets 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 dise ase-free and overall survival of patients with various malignancies un dergoing autotransplantation.