High level of retrovirus-mediated gene transfer into dendritic cells derived from cord blood and mobilized peripheral blood CD34(+) cells

Dendritic cells (DCs), the most potent antigen-presenting cells, can be gen erated from CD34(+) hematopoietic stem cells and used for generating therap eutic immune responses. To develop immunotherapy protocols based on genetic ally modified DCs, we have investigated the conditions for high-level trans duction of a large amount of CD34(+)-derived DCs. Thus, we have used an eff icient and clinically applicable protocol for the retroviral transduction o f cord blood (CB) or mobilized peripheral blood (MPB) CD34(+) cells based o n infection with gibbon ape leukemia virus (GALV)-pseudotyped retroviral ve ctors carrying the nls-LacZ reporter gene. Infected cells have been subsequ ently cultured under conditions allowing their dendritic differentiation. T he results show that using a growth factor combination including granulocyt e-macrophage colony-stimulating factor plus tumor necrosis factor alpha plu s interleukin 4 plus stem cell factor plus Flt3 ligand, more than 70% of DC s derived from CB or MPB CD34(+) cells can be transduced, Semiquantitative PCR indicates that at least two proviral copies per cell were detected. Tra nsduced DCs retain normal immunophenotype and potent T cell stimulatory cap acity. Finally, by using a semisolid methylcellulose assay for dendritic pr ogenitors (CFU-DCs), we show that more than 90% of CFU-DCs can be transduce d, Such a highly efficient retrovirus-mediated gene transfer into CD34(+)-d erived DCs makes it possible to envision the use of this methodology in cli nical trials.