Histone H1-mediated transfection: serum inhibition can be overcome by Ca2+ ions.
OBJECTIVE
One of the drawbacks of polycationic and cationic liposomal gene transfer is its sensitivity to serum. Gene therapy requires the transfectant-DNA complex to be resistant to serum as well as blood. Since Ca2+ has proved 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 ECV 304 human endothelial cells using H1 histone and DOSPER as transfectants in the presence of 0-100% fetal calf serum.


RESULTS
H1-and DOSPER-mediated transfection was found to be inhibited by serum above the concentration of 10%. If 2 mM Ca2+ or 2 mM Ca2+/0.1 mM chloroquine was included in the culture medium which replace the transfection mixture 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 endosomal escape. Ca2+ in the form of nascent calcium phosphate microprecipitates and other lysosomolytical agents facilitate endosomal/lysosomal release by their fusigenic and membranolytic activity.
Haberland
A
A
Franz Volhard Clinic at the Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.
Knaus
T
T
Zaitsev
S V
SV
Buchberger
B
B
Lun
A
A
Haller
H
H
Böttger
M
M
D001798:Blood Proteins / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002461:Cell Line, Transformed; D003470:Culture Media / Q000494:pharmacology; D004247:DNA / Q000493:pharmacokinetics; D004730:Endothelium, Vascular / Q000166:cytology; D015870:Gene Expression / Q000187:drug effects; D006657:Histones / Q000235:genetics* / Q000493:pharmacokinetics*; D006801:Humans; D008081:Liposomes; D014162:Transfection / Q000187:drug effects* / Q000379:methods*; D019076:Transgenes / Q000235:genetics; D014471:Umbilical Veins / Q000166:cytology
D001798:Blood Proteins; D003470:Culture Media; D006657:Histones; D008081:Liposomes; D004247:DNA; D002118:Calcium