Abstract
Purpose. One of the drawbacks of polycationic and cationic liposomalgene transfer is its sensitivity to serum. Gene therapy requires thetransfectant-DNA complex to be resistant to serum as well as blood.Since Ca2+ has proved to be an efficient cofactor of polycationic genetransfer, we decided to investigate its effects on transfection in thepresence of serum.
Methods. We studied transgene expression of luciferase gene (pCMVLuc) on ECV 304 human endothelial cells using H1 histone andDOSPER as transfectants in the presence of 0-100% fetal calf serum.
Results. H1-and DOSPER-mediated transfection was found to beinhibited by serum above the concentration of 10%. If 2 mM Ca2+ or2 mM Ca2+/0.1 mM chloroquine was included in the culture mediumwhich replace the transfection mixture and was left on the cells for24 hours postincubation, the inhibiting effect of even 100% serumwas overcome.
Conclusions. A high serum level does not interfere with binding anduptake of H1- and DOSPER-DNA complexes, but inhibits subsequentsteps such as endosomal escape. Ca2+ in the form of nascent calciumphosphate microprecipitates and other lysosomolytical agents facilitateendosomal/lysosomal release by their fusigenic and membranolyticactivity.
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Haberland, A., Knaus, T., Zaitsev, S.V. et al. Histone H1-Mediated Transfection: Serum Inhibition Can Be Overcome by Ca2+ Ions. Pharm Res 17, 229–235 (2000). https://doi.org/10.1023/A:1007581700996
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DOI: https://doi.org/10.1023/A:1007581700996