Abstract
Injection of a recombinant adenovirus expressing human bilirubin-UGT1 (Ad-hBUGT1) (3 x 10(9) plaque-forming units (pfu) intravenously) in adult bilirubin-UDP-glucuronosyltransferase-1 (BUGT1)-deficient Gunn rats resulted in biliary excretion of bilirubin glucuronides and a 70% reduction of serum bilirubin levels. However, the effect was transient, and host humoral and cellular immune response prevented transgene expression after subsequent injections. To determine whether injection during the neonatal period would tolerize the host to the recombinant virus, we injected 1 x 10(8) pfu of Ad-hBUGT1 or Ad-LacZ (a recombinant adenovirus expressing Escherichia coli beta-galactosidase) into 1-3-day-old Gunn rats. Two subsequent injections (3 x 10(9) pfu) were given 56 and 112 days after the initial injection. Injection of Ad-BUGT1, but not Ad-LacZ, reduced serum bilirubin by 70-76% of the levels in untreated pups (9 +/- 1.3 mg/dl), followed by a gradual increase to 3.25 +/- 0.3 mg/dl in 56 days; similar or greater reductions occurred after the second and third injection. Serum neutralizing antibody titer and cytotoxic lymphocyte activity against adenovirus-infected hepatocytes were low or undetectable. Thus, tolerization by injection of the virus during the neonatal period permits long term gene therapy by repeated injection of the virus.
Highlights
Injection of a recombinant adenovirus expressing human bilirubin-UGT1 (Ad-hBUGT1) (3 ؋ 109 plaque-forming units intravenously) in adult bilirubin-UDPglucuronosyltransferase-1 (BUGT1)-deficient Gunn rats resulted in biliary excretion of bilirubin glucuronides and a 70% reduction of serum bilirubin levels
To determine whether the rats that are tolerized to recombinant adenoviruses are capable of mounting an immune response to wild-type adenoviruses, two Gunn rats that were tolerized to Ad hBUGT1 by injection during the newborn period were injected with wild-type adenovirus 5 after the third AdhBUGT1 injection
A moderate fluctuation of serum bilirubin levels in Gunn rats can occur as a result of a variety of metabolic factors, direct demonstration of bilirubin glucuronide excretion in bile in our experiments constitutes an unequivocal evidence for glucuronidation of bilirubin
Summary
Homozygous Gunn rats were bred in our colony at the Albert Einstein College of Medicine, maintained on standard laboratory chow, and kept in 12-h light/dark cycles. The recombinant adenovirus Ad-hBUGT1 was generated by cotransfection of pAd-BglIIhBUGT1 and pJM17 into 293 cells as described [18]. Group A were Gunn rats that received three intravenous injections Ad-hBUGT1 at the ages of 1–3, 56, and 112 days, respectively. Group B Gunn rats received Ad-hBUGT1 injection intravenously as newborns, and the second and third injections, at ages 56 and 112 days, respectively, into the portal vein. Group C were newborn Gunn rats injected with Ad-LacZ intravenously. Group D were adult Gunn rats injected intravenously with Ad-hBUGT1. Group E were newborn normal RHA rats injected intravenously with Ad-hBUGT1 virus. Group F included newborn Gunn rats injected intravenously with Ad-hBUGT1 for the first injection, followed by Ad-LacZ intravenously for the second injection. Bile Pigment Analysis—For definitive evaluation of bilirubin glucuronidation, selected rat were provided with a polyethylene bile duct cannula and pigments excreted in bile were analyzed by HPLC using a Bondapak C-18 column (Millipore-Waters, Milford, MA) as described [20]
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