CATIONIC LIPID-MEDIATED TRANSFER OF THE HIL-10 GENE PROLONGS SURVIVALOF ALLOGENEIC HEPATOCYTES IN NAGASE ANALBUMINEMIC RATS

Gene transfer techniques can be used as a drug delivery system to achi eve local immunosuppression. We performed a series of experiments to i dentify the cationic lipid that most efficiently transfects isolated, cultured, rat hepatocytes; to optimize conditions for efficient transf ection; to determine the duration of gene expression in vitro; and fin ally, 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 mu g/35 mm culture plate seeded with 2. 5 x 10(5) rat hepatocytes. When transfected as described above, cultur ed hepatocytes expressed the hIL-10 gene for approximately 14 days. Ac cordingly, Nagase rats transplanted with 4 x 10(7) DOTAP-hIL-10 transf ected, allogeneic hepatocytes had an abrupt rise in serum albumin leve ls that peaked within 7 days of the transplant, decreased abruptly aft er 15 days, and approached baseline by day 40. In contrast, control an imals had a smaller albumin peak. that returned to baseline within 10 days (P<0.01). In all animals, serum hIL-10 levels were undetectable w hen tested. We conclude that DOTAP is the best cationic lipid for tran sfection of cultured rat hepatocytes. Furthermore, hIL-10 transfected hepatocytes have a prolonged survival in an allogeneic horst which is probably limited by toss of gene expression. Further studies using oth er vectors capable of prolonged gene expression will help determine if indefinite hlL-10 gene expression leads to indefinite graft survival.