UMBILICAL-CORD BLOOD-CELL TRANSDUCTION BY RETROVIRAL VECTORS - PRECLINICAL STUDIES TO OPTIMIZE GENE-TRANSFER

Human umbilical cord blood (UCB) can be a source of hematopoietic stem cells for gene therapy, as an alternative to allogeneic bone marrow t ransplantation, for the treatment of a number of genetic diseases. To determine conditions that yield maximal gene transfer into UCB progeni tor cells, we examined a number of variables. We used cell-free retrov iral vector supernatants that convey neomycin (G418) resistance and me asured the percentage of G418-resistant progenitor-derived colonies. A dding retroviral supernatant to the UCB cells in basal medium once a d ay for 3 days produced a threefold increase of G418-resistant colonies (9.8%) compared to a single exposure to supernatant (3.1%). To establ ish whether recombinant human growth factors are beneficial during tra nsduction, the presence of interleukin-3 (IL-3), IL-6, and mast cell g rowth factor (MGF, a c-kit ligand) were compared in different combinat ions. Inclusion of the three factors together caused a threefold incre ase of gene transfer (30.4%) compared to transduction in basal medium. When the UCB cells were precultured in medium containing IL-3, IL-6, and MGF for 3 days before addition of the retroviral supernatant on da ys 4, 5, and 6, the average extent of gene transfer was 21.8%, compare d with an average of 34.4% when UCB cells were transduced on days 1, 2 , and 3. The presence of marrow stroma during the transduction of the UCB cells did not further increase gene transfer. We conclude that UCB progenitor cells can be efficiently transduced with the use of recomb inant human growth factors IL-3, IL-6, and MGF and may be a suitable s ource for gene therapy.