Gene transfer by retrpvirus vectors into human T-lymphocytes has been applied for correcting genetic diseases and modulating immune functions. The main limit of this procedure is the low efficiency of transaction that can be achieved by retrovirus vectors. In an attempt to investigate alternative strategies for the transduction of human T lymphocytes, we have evaluated as a potential vector a recombinant adenovirus (Ad-MFG-AP), carrying a modified membrane-exposed alkaline phosphatase (AP), as reporter gene. The transgene expression was evaluated by indirect immunofluorescence flow-cytometry assay with an anti-alkaline phosphatase antibody. CD3+ cells were selected from the buffy-coat of healty donors (n=6) by immune-magnetic technique (median CD3+ cell purity= 95±1%). 0.5x106 selected CD3+ cells were incubated in 10% human serum RPMI with IL2 alone, or in combination with IL4. IL7. IL12, FLT-3 Ligand for 7 days prior to Ad-MFG-AP infection for 1 hr at the optimal virus:target ratio of 500:1. After infection, the lymphocytes were incubated with 1L2 for 48 hrs before evaluation of transgene expression. The best results were achieved incubating CD3+ cells with IL2+IL12 or with IL2+IL7. To increase the percentage of AdMFG-AP infected cells, we have utilized a cationic lipid, Lipofectamine, that mediates the attachment of adenovirus particles to the cell surface. Lipofectamine/Ad-MFG-AP complexes were formed by mixing 15 uL of Lipofectamine (30 ng) with r-Ad-MFG-AP at a MOI of 500 in 0.5 mL serum-free RPMI 1640. The mixture was incubated at room temperature for 30 min and then added to target cells. The results are illustrated above: % AP+ Cells Cytokines Ad-MFG-AP Lipof/Ad-MFG-AP IL BE TE IL2+FLT-3 7±5 ND IL2+IL4 6±4 ND IL2+IL7 10±1 34±5 IL2+IL12 1511 30±2 In conclusion, a 7-day culture with IL2 and IL12 or IL7 represents a powerful stimulation to adenovirus/Lipofectamme mediated gene transduction in clinically applicable conditions.
|Number of pages||1|
|Publication status||Published - 1998|
ASJC Scopus subject areas
- Cancer Research
- Cell Biology