Recombinant adenoviral vector-LipofectAMINE complex for gene transduction into human T lymphocytes

We have evaluated, as a vector for gene transfer into human T lymphocytes, a recombinant adenovirus (rAd-MFG-AP) carrying a modified, membrane-exposed , alkaline phosphatase (AP) as reporter gene. CD3(+) cells were selected fr om the buffy coat of healthy donors by the immunomagnetic technique. The po sitive cell population, comprising 96 +/- 2% CD3(+) cells, was cultured wit h clinical-grade cytokine(s) for 3-7 days prior to rAd-MFG-AP transduction and the transgene expression was evaluated 48 hr later by indirect immunofl uorescence flow cytometry assay with an anti-alkaline phosphatase antibody, The best efficiency of transduction was achieved on incubation of CD3(+) c ells with IL-2 plus either IL-12 (AP(+) cells, 12 +/- 3%) or IL-7 (AP(+) ce lls, 11 +/- 3%). To increase further the efficiency of transduction, we hav e combined LipofectAMINE and rAd-MFG-AP with the aim to enhance the uptake of viral particles into the target cells. The percentage of CD3(+) cells tr ansduced by rAd-MFG-AP-LipofectAMINE complex was 24 +/- 4% (range, 20-35%) after incubation with IL-2 plus IL-7 and 22 +/- 4% (range, 18-32%) after in cubation with II-2 plus IL-12, Forty-eight hours after the incubation with rAd-MFG-AP, the transduced T lymphocytes were subjected to fluorescence-act ivated cell sorting and fractionated into AP(+) and AP(-) cell subpopulatio ns. The AP(+) cell fraction, comprising 96.8% of AP(+) cells, was evaluated by FACScan analysis for T lymphocyte surface antigens, The immunophenotypi ng of the transduced T lymphocytes has shown that there was not a particula r subtype of T lymphocytes more susceptible to rAd-MFG-AP transduction, In addition, the transgene expression did not modify T lymphocyte functions, a s demonstrated by results obtained by cytotoxicity assay before and after r Ad-MFG-AP-LipofectAMINE complex transduction, In conclusion, human T lympho cytes can be efficiently transduced, under clinically applicable conditions , by adenovirus-lipofectAMINE complex after 7 days of culture with IL-2 and IL-12 or IL-7.