COMPLETE CORRECTION OF HYPERBILIRUBINEMIA IN THE GUNN RAT MODEL OF CRIGLER-NAJJAR SYNDROME TYPE-I FOLLOWING TRANSIENT IN-VIVO ADENOVIRUS-MEDIATED EXPRESSION OF HUMAN BILIRUBIN UDP-GLUCURONOSYLTRANSFERASE

Recombinant adenoviral vectors are useful for the in vivo expression o f genes in hepatocytes. Adenoviral vectors deleted in E1a, E1b and E3b were constructed and used to study in vivo expression of the major hu man bilirubin UDP-glucuronosyltransferase isoform (HUG Br1) under the transcriptional control of the cytomegalovirus (CMV) immediate-early p romoter-enhancer (H5.010CMVhugBr1). As a control, a recombinant adenov iral vector containing the beta-galactosidase reporter gene driven by the CMV promoter-enhancer was employed (H5.010CMVlacZ). Recombinant vi rus was expanded following exposure to E1 transcomplementing (293) cel ls and concentrated to a titer of approximately 10(13) particles per m illiliter. A rat model for Crigler-Najjar syndrome type I deficient in HUG Br1 (ie the Gunn rat) was injected with 5 x 10(9) plaque-forming units (p.f.u.) via the portal vein of either H5.010CMVhugBr1 or H5.010 CMVlacZ. Rats from each set were killed at 3 days, 11 days and 22 days after infusion. Liver total cellular DNA, RNA and protein were analyz ed for the transgene and the transgene product at the specified times. Analysis of livers by Southern blot hybridization demonstrates sequen ce-specific hybridization to adenoviral vector DNA, and Northern blot hybridization demonstrates sequence-specific hybridization to transgen e-derived RNA. DNA levels peak at approximately one copy number at 3 d ays and decline over 22 days. RNA and Western blot analyses demonstrat e overexpression of message and protein at 3 days, declining over 22 d ays. In vitro functional assay for bilirubin glucuronosyltransferase a ctivity demonstrates overexpression of bilirubin UDP-glucuronosyltrans ferase function. In situ hybridization of frozen sections to detect ex pressed mRNA using beta-galactosidase derived S-35-labeled riboprobes demonstrates adenovirus-derived transgene expression in hepatocytes. S ignificant drops in serum bilirubin levels were noted following expres sion of HUG Br1 but not beta-galactosidase. The drop in serum bilirubi n correlates with the appearance of bilirubin glucuronides in bile. In summary, recombinant adenoviral vectors were used to demonstrate in v ivo complementation of the genetic defect in Gunn rat livers with the HUG Br1 cDNA leading to a resolution of hyperbilirubinemia lasting for approximately 7 weeks. These studies suggest that delivery of the HUG Br1 cDNA might provide a reasonable therapeutic benefit for Crigler-N ajjar syndrome type I patients as safe and efficacious gene delivery s ystems are developed.