Large-scale feasibility of gene transduction into human CD34(+) cell-derived dendritic cells by adenoviral/polycation complex

With a view to using multiple injections of anticancer dendritic cell (DC)- based vaccines, we evaluated the feasibility of the adenoviral transduction of large amounts of human CD34(+) cell-derived DCs, and analysed the persi stence of the transgene expression and the integrity of DC functional activ ity after the transduction/cryopreservation procedures. Mature DCs generate d from highly enriched human CD34(+) cells were transduced by a recombinant adenovirus (rAd-MFG) that carried a modified, membrane-exposed, alkaline p hosphatase (AP) sequence as the reporter gene. Cationic lipids such as Lipo fectAmine or poly-L-lysine were mixed with the viral particles before the t ransduction of the target cells. The highest transduction efficiency was ob tained at a multiplicity of infection (MOI) rate of 500 (AP + DCs: 50 +/- 2 %, viability = 95%) under both small- and large-scale conditions. The addit ion of poly-L-lysine or LipofectAmine increased the percentage of transduce d cells at an MOI of 500 (CD1a(+)/AP(+) cells = 85 +/- 3% and 80 +/- 2% res pectively). Polycations made it possible to reduce the amounts of viral par ticles, with high efficiency of transduction being achieved at a MOI of 100 with 10 mug/ml poly-L-lysine (CD1a(+)/AP(+): 68 +/- 9%) or 30 mug/ml Lipof ectAmine (CD1a(+)/AP(+): 60 +/- 7%). Evaluation of the immunophenotype of t he transduced DCs showed that the lack of a DC subpopulation was more susce ptible to adenoviral transduction. Cryopreservation of transduced DCs did n ot modify the viability or percentage of AP(+) cells that maintain antigen- presenting cell (APC) functions. These findings indicate the efficacy of th is method for the transduction of large amounts of CD34(+) cell-derived DCs using small quantities of adenoviral vector mixed with polycations. Cryopr eservation of transduced DCs did not damage their viability or APC function s, thus making it possible to plan multiple injections of engineered DC-bas ed vaccines.