Recombinant adenoviral vectors are useful for the in vivo expression of genes in hepatocytes. Adenoviral vectors deleted in E1a, E1b, and E3b were constructed and used to study in vivo expression of the major human
bilirubin UDP-glucuronosyltransferase isoform (HUG Br1) under the transcriptional control of the cytomegalovirus (CMV) immediate-early promoter-enhancer (H5.010CMV hugBr1). As a control, a recombinant adenoviral vector containing the
beta-galactosidase reporter gene driven by the CMV promoter-enhancer was employed (H5. 010CMVlacZ). Recombinant virus was expanded following exposure to E1 transcomplementing (293) cells and concentrated to t titer of approximately 10(13) particles per milliliter. 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. 010CMVlacZ. Rats from each set were killed at 3 days, 11 days and 22 days after infusion. Liver total cellular
DNA,
RNA and
protein were analyzed for the transgene and the transgene product at the specified times. Analysis of livers by Southern blot hybridization demonstrates sequence-specific hybridization to adenoviral vector
DNA, and Northern blot hybridization demonstrates sequence-specific hybridization to transgene-derived
RNA.
DNA levels peak at approximately one copy number at 3 days and decline over 22 days.
RNA and Western blot analyses demonstrate overexpression of message and
protein at 3 days, declining over 22 days. In virto functional assay for
bilirubin glucuronosyl-
transferase activity demonstrates overexpression of
bilirubin UDP-glucurosyltransferase function. In situ hybridization of frozen sections to detect expressed
mRNA using beta-galactosidasederived 35S-labeled riboprobes demonstrates adenovirus-derived transgene expression in hepatocytes. Significant drops in serum
bilirubin levels were noted following expression of HUG Br1 but not
beta-galactosidase. The drop in serum
bilirubin correlates with the appearance of
bilirubin glucuronides in bile. In summary, recombinant adenoviral vectors were used to demonstrate in vivo complementation of the genetic defect in Gunn rat livers with the HUG Br1
cDNA leading to a resolution of
hyperbilirubinemia lasting approximately 7 weeks. These studies suggest that delivery of the HUG Br1
cDNA might provide a reasonable therapeutic benefit for
Crigler-Najjar syndrome type I patients, as safe and efficacious gene delivery systems are developed.