Enhancement of Nonviral Gene Transfer to Endothelial Cells Using Lipofection of Histone-Complexed DNA

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Abstract
Arterial gene transfer represents a novel approach for the treatment of a variety of vascular diseases. Lipofectamine (DOSPA/DOPE)-mediated transfer of pCMVβgal (7.2 kbp) to subconfluent bovine aortic endothelial cells (BAEC) resulted in a transfection efficiency of 5.7 ± 2.35% of cells staining positive for β-galactosidase for a 10:1 ratio by weight of lipid/plasmid. Using histones up to 1 histone per 5 bp increased efficiency over 3-fold higher to a level of 18.2 ± 1.8%. In confluent BAEC, which are more difficult to lipofect, the use of histones increased the transfection efficiency over 10-fold to a level of 4.5%. Histones form a tight compact structure with the DNA and also protect DNA from DNase degradation. Using histones as a compacting agent while lowering the amount of lipofectamine resulted in a large drop in transfection efficiency, demonstrating that a critical liposome/plasmid ratio had to be maintained. When histones were not used, posttransfection treatment of endothelial cells with 100 µM chloroquine, which helps in disruption of the early endosome by increasing endosome pH, caused a 2.5-fold increase over transfection levels without chloroquine. However, chloroquine provided no benefit when plasmids were histone compacted. Thus, the use of chloroquine is not required. Since the improvements in transfection efficiency with histones (via improved DNase protection) and with chloroquine (via improved endocytotic escape) were not additive, the final rate limit to transfection may be plasmid entry into the nucleus. Ligands that facilitate receptor binding or fusigenic agents that promote endosome escape can aid in lipofection. Still, such approaches will not fully overcome the limits on efficiency that exist for nondividing endothelial cells in vivo.

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