Crigler-Najjar syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by a deficiency of uridine diphospho-glucuronosyl transferase 1A1. Current therapy relies on phototherapy to prevent kernicterus, but liver transplantation presently is the only permanent cure. Gene therapy is a potential alternative, and recent work has shown that helper-dependent adenoviral (HD-Ad) vectors, devoid of all viral coding sequences, induce prolonged transgene expression and exhibit significantly less chronic toxicity than early-generation Ad vectors. We used a HD-Ad vector to achieve liver-restricted expression of human uridine diphospho-glucuronosyl transferase 1A1 in the Gunn rat, a model of the human disorder. Total plasma bilirubin levels were reduced from >5.0 mg͞dl to Ͻ Ͻ1.4 mg͞dl for >2 yr after a single i.v. administration of vector expressing the therapeutic transgene at a dose of 3 ؋ 10 12 viral particles per kg. HPLC analysis of bile from treated rats showed the presence of bilirubin glucuronides at normal WT levels >2 yr after one injection of vector, and i.v. injection of bilirubins III␣ and XIII␣ in the same animals revealed excess bilirubin-conjugating capacity. There was no significant elevation of liver enzymes (alanine aminotransferase) and only transient, moderate thrombocytopenia after injection of the vector. A clinically significant reduction in serum bilirubin was observed with a dose as low as 6 ؋ 10 11 viral particles per kg. We conclude that complete, long-term correction of hyperbilirubinemia in the Gunn rat model of Crigler-Najjar syndrome can be achieved with one injection of HD-Ad vector and negligible chronic toxicity.adenovirus ͉ bilirubin ͉ gene therapy

Bilirubin, the yellow pigment of jaundice, is a linear tetrapyrrole with a methylene group at its center, C(10), a position of crucial importance to its conformation and metabolism. The presence of the central methylene group allows the bilirubin to fold into an intramolecularly hydrogen-bonded conformation. This paper describes the first synthesis of a bilirubin analogue with an oxo group at C(10). The change from CH2 to CO, from sp3 to sp2, is designed to stress the molecule at its hinge and relax its conformation. Such compounds have been suggested as potential oxidative metabolites of bilirubin in vivo. 10-Oxo-mesobilirubin-XIIIα (1) is a red crystalline solid, unlike its parent, mesobilirubin-XIIIα, which is a bright yellow solid. It is surprisingly polar, relative to the parent, yet it does not exhibit a significantly larger bicarbonate/chloroform partition coefficient. Like the parent, 1 appears to adopt an intramolecularly hydrogen-bonded ridge-tile-like conformation. In normal rats, 1 is metabolized to acylglucuronides, which are secreted into bile, but a portion of the administered dose is secreted into bile intact. In mutant rats (Gunn rats) lacking bilirubin glucuronyl transferase, 1 was excreted efficiently in bile in unchanged form, unlike the parent with a methylene group at C(10). Thus, introduction of the oxygen function at C(10) has little effect on hepatic uptake but a dramatic effect on canalicular secretion into bile.