This study was initiated in order to improve the transduction efficiency of adenoviral vectors (Ad) in human CD34(+) hematopoietic progenitor cells. CD34(+)-cells (0.5-1 x 105) from cord or mobilized peripheral blood were incubated with TNFa (0.01 to 1 mg/ml) for 2 hours. After removal of TNF, the cells were infected with an adenoviral vector encoding for GFP (MOI 500, 18 hours). Two days later, the viable cells were counted and analyzed for GFP and CD34 by flow cytometry. Plating efficiencies pre- and post-transduction were evaluated by methylcellulose assay. To visualize vectorial trafficking, CD34(+) cells were incubated with fluorophore-conjugated Ad. In a dose dependent manner, pretreatment with TNFa increased transduction efficiency more than 2fold (39.2 versus 15.5%) and improved survival of CD34(+)-cells resulting in a 6fold increase of CD34(+)GFP(+)-cells (24.4 versus 4.2 x 103), which was maintained for five days. Induction of apoptosis was excluded by Annexin and propidium iodide. Plating efficiency, especially CFU-GM formation was enhanced by TNF. Analysis of viral binding demonstrated a significantly higher incidence of local concentrations of Ad along the cell surface ( caps ) in infected cells of the TNFtreated group (8±2.4% versus 18.1±5%; p< 0.05). No difference was found in the relative number of infected cells. Time course experiments showed that the effect of TNF could be observed with initial incubation times as short as 10 minutes. FACS-analysis did not indicate a significant increase in adenoviral receptors (CAR, av-integrins). Short-term incubation of CD34(+)-cells with TNFa before adenoviral infection improves transduction and plating efficiency as well as cell survival. This finding correlated with increased Ad capping at the cell surface but not Ad receptor expression suggesting that Ad trafficking may be altered.
|Issue number||11 PART I|
|Publication status||Published - 1 Dec 2000|
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