Cationic lipid-mediated transfer of the hIL-10 gene prolongs survival of allogeneic hepatocytes in Nagase analbuminemic rats.
Gene transfer techniques can be used as a drug delivery system to achieve local immunosuppression. We performed a series of experiments to identify the cationic lipid that most efficiently transfects isolated, cultured, rat hepatocytes; to optimize conditions for efficient transfection; to determine the duration of gene expression in vitro; and finally, to determine the survival of allogeneic hepatocytes transplanted into Nagase rats. Our results suggest that DOTAP is the best cationic lipid for transfection of cultured rat hepatocytes. In addition, the following conditions appear to optimize transfection efficiency: a DNA:DOTAP ratio of 1:6; a 24 exposure time of the hepatocytes to the DNA-DOTAP complex; a DNA dose of 4 microg/35 mm culture plate seeded with 2.5x10(5) rat hepatocytes. When transfected as described above, cultured hepatocytes expressed the hIL-10 gene for approximately 14 days. Accordingly, Nagase rats transplanted with 4x10(7) DOTAP-hIL-10 transfected, allogeneic hepatocytes had an abrupt rise in serum albumin levels that peaked within 7 days of the transplant, decreased abruptly after 15 days, and approached baseline by day 40. In contrast, control animals had a smaller albumin peak that returned to baseline within 10 days (P<0.01). In all animals, serum hIL-10 levels were undetectable when tested. We conclude that DOTAP is the best cationic lipid for transfection of cultured rat hepatocytes. Furthermore, hIL-10 transfected hepatocytes have a prolonged survival in an allogeneic host which is probably limited by loss of gene expression. Further studies using other vectors capable of prolonged gene expression will help determine if indefinite hIL-10 gene expression leads to indefinite graft survival.
Fabrega
A J
AJ
Department of Surgery, Department of Medicine and Howard Hughes Medical Institute, University of Iowa, Iowa City 52242, USA.
Fasbender
A J
AJ
Struble
S
S
Zabner
J
J
D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006085:Graft Survival / Q000187:drug effects; D006801:Humans; D007108:Immune Tolerance / Q000187:drug effects; D016753:Interleukin-10 / Q000235:genetics*; D008055:Lipids / Q000235:genetics; D008099:Liver / Q000166:cytology*; D016031:Liver Transplantation / Q000276:immunology*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D051381:Rats; D011914:Rats, Inbred BN; D014162:Transfection / Q000187:drug effects
D005229:Fatty Acids, Monounsaturated; D008055:Lipids; D000644:Quaternary Ammonium Compounds; D016753:Interleukin-10; D004247:DNA; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane