Pre-clinical evaluation of an in vitro selection protocol for the enrichment of transduced CD34(+) cell-derived human dendritic cells

The efficient genetic modification of CD34(+) cell-derived dendritic cells (DC) will provide a significant. advancement towards the development of imm unotherapy protocols for cancer, autoimmune disorders and infectious diseas es. Recent reports have described the transduction of CD34(+) cells via ret rovirus- and lentivirus-based gene transfer vectors and subsequent differen tiation into functional DC. Since there is significant apprehension regardi ng the clinical uses of HIV-based vectors, in this report, we compare a mur ine leukemia virus (MLV)- and a human immunodeficiency virus (HIV)-based bi cistronic vector for gene transfer into human CD34(+) cells and subsequent differentiation into mature DC. Each vector expressed both EGFP and the dom inant selectable marker DHFRL22Y allowing for the enrichment of marked cell s in the presence of the antifolate drug trimetrexate (TMTX). Both MLV-base d and HIV-based vectors efficiently transduced cytokine mobilized human per ipheral blood CD34(+) cells. However, in vitro expansion and differentiatio n in the presence of GM-CSF, TNF-alpha, Flt-3L, SCF and IL-4 resulted in a reduction in the percentage of DC expressing the transgene. Selection with TMTX during differentiation increased the percentage of marked DC, resultin g in up to 79% (MLV vector) and up to 94% (lentivirus-vector) transduced ce lls expressing EGFP without loss of DC phenotype. Thus, MLV-based vectors a nd in vitro selection of transduced human DC show great promise for immunot herapy protocols.