Purpose. One of the drawbacks of polycationic and cationic liposomal gene t
ransfer is its sensitivity to serum. Gene therapy requires the transfectant
-DNA complex to be resistant to serum as well as blood. Since Ca2+ has prov
ed to be an efficient cofactor of polycationic gene transfer, we decided to
investigate its effects on transfection in the presence of serum.
Methods. We studied transgene expression of luciferase gene (pCMV Luc) on E
CV 304 human endothelial cells using I-Il histone and DOSPER as transfectan
ts in the presence of 0-100% fetal calf serum.
Results. Hi-and DOSPER-mediated transfection was found to be inhibited by s
erum above the concentration of 10%. If 2 mM Ca2+ or 2 mM Ca2+/0.1 mM chlor
oquine was included in the culture medium which replace the transfection mi
xture and was left on the cells for 24 hours postincubation, the inhibiting
effect of even 100% serum was overcome.
Conclusions. A high serum level does not interfere with binding and uptake
of H1- and DOSPER-DNA complexes, but inhibits subsequent steps such as endo
somal escape. Ca2+ in the form of nascent calcium phosphate microprecipitat
es and other lysosomolytical agents facilitate endosomal/lysosomal release
by their fusigenic and membranolytic activity.