Strong, long-term transgene expression in rat liver using chicken beta-actin promoter associated with cytomegalovirus immediate-early enhancer (CAG promoter)

For successful gene therapy in hepatic enzyme deficiencies, it is essential to use promoters that can maintain strong transcriptional activity for the long term in the liver. Using Gunn rats, a model animal for Crigler-Najjar syndrome type I, the long-term transcriptional function of the CAG promote r (a combination of chicken beta -actin promoter and cytomegalovirus immedi ate-early enhancer) was evaluated in the rat liver. We constructed a plasmi d pCAGGHUGT, containing expression cassettes of human bilirubin UDP-glucuro nosyltransferase (BUGT) and hygromycin phosphotransferase, under the contro l of the CAG promoter and murine phosphoglycerate kinase promoter, respecti vely. Conditionally immortalized Gunn rat hepatocytes (IGRH), which had bee n established using mutant SV40 large T antigen (T-TS), were transfected wi th pCAGGHUGT. A stably transfected clone IGRHUGT, expressing a high level o f BUGT, was obtained after selection with hygromycin. At 33 degreesC, the c ells doubled in number in approximately 72 h; however, at 37 degreesC, cell proliferation stopped, indicating that the characteristic of temperature-d ependent proliferation was retained in this clone. Ten million cells were i njected into the spleen of syngeneic Gunn rats five times at 10-day interva ls. Serum bilirubin levels were reduced by 45-50% at 70 days after the firs t transplantation and remained so throughout the duration of the study (120 days). These results suggested that the CAG promoter was able to maintain strong transcriptional activity in rat liver for at least 120 days.