Critical assessment of the nuclear import of plasmid during cationic lipid-mediated gene transfer

Background Cationic lipid-mediated gene transfer is a promising approach fo r gene therapy. However, despite the significant amount of lipoplexes inter nalized by target cells, transgene expression remains too low. Obstacles to nuclear accumulation of plasmid DNA include: the passage of DNA across the cellular membrane, the dismantling of nucleolipidic particles in the cytop lasm and the nuclear import of plasmid DNA. The purpose of the present stud y was to evaluate the impact of cell status on cationic lipid-mediated tran sfer. Methods Cells were either growth-arrested (by aphidicolin) or synchronized (by a classical double-thymidine block protocol) and cationic lipid-mediate d transfection of these cells was evaluated. For the study of the nuclear i mport of plasmid DNA, two techniques were developed: microinjection of plas mid DNA into intact cells, and the use of cells permeabilized with digitoni n. Results When CV-1 cells were growth-arrested by aphidicolin, cationic lipid -mediated gene transfer was inhibited. Hela cells were synchronized and inc ubated with lipoplexes at different times after release of the block. Gene expression was greatly enhanced when cells underwent mitosis. When transfec tion was performed during the early period after block release, when fewer than 5% of the cells had divided, gene expression was carefully quantified and could be attributed to cells that escaped cell cycle block. However, by direct analysis of nuclear import of GFP-coding plasmid using cytoplasmic microinjection, GFP expression could be detected in a few cells that had no t divided. Conclusions Cationic lipid-mediated gene transfer efficiency increased when cells underwent mitosis. However, when cells did not divide, gene transfer was not completely abolished. Nuclear import of plasmid was greatly facili tated by a mitotic event. In non-mitotic cells, nuclear envelope crossing b y plasmid DNA could be detected but was a very rare event. Copyright (C) 20 01 John Wiley & Sons, Ltd.