Gene Therapy with Bilirubin-UDP-Glucuronosyltransferase in the Gunn Rat Model of Crigler-Najjar Syndrome Type 1

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Abstract
Crigler-Najjar syndrome type 1 (CN type 1) is an autosomal recessive disorder characterized by nonhemolytic jaundice resulting from mutations to the gene encoding bilirubin-UDP-glucuronosyltransferase (UDPGT). The Gunn rat is an accurate animal model of this disease because the bilirubin-UDPGT gene in this strain carries a premature stop codon. The primary objective of this study was to complement this deficiency in vivo using liver-directed gene therapy. The efficiency of adenovirus type 5 (Ad5)-mediated gene transfer to the neonatal rat liver was first assessed by intravenous (i.v.) injection of an Ad5 vector carrying a nuclear-localized LacZ gene. An Ad5 vector expressing the cDNA encoding human bilirubin-UDPGT (Ad5/CMV/hUG-Br1) was then generated and injected i.v. into neonatal Gunn rats. Plasma samples were collected and bilirubin levels were determined at regular intervals. Although the mean level of bilirubin in homozygous Gunn rats 1–2 days after birth was already 14.5-fold higher than that of heterozygous siblings, treatment with Ad5/CMV/hUG-Br1 reduced plasma bilirubin to normal levels within 1 week. Plasma bilirubin in the treated homozygous rats remained normal for 4 weeks before gradually climbing to intermediate levels that were approximately half that of untreated homozygotes by 12 weeks. Administration of Ad5-mediated gene therapy to neonatal Gunn rats effectively complemented the deficiency in bilirubin-UDPGT, resulting in substantial reductions in plasma bilirubin over a 3-month period. The efficacy of Ad5-mediated gene therapy in neonates suggests that this approach might be effective against other hepatic disorders, including autosomal recessive deficiencies in lipid metabolism and vascular homeostasis. Crigler-Najjar syndrome type 1 (CN type 1) is an autosomal recessive disorder afflicting liver metabolism that results from well-defined genetic lesions to the gene encoding bilirubin-UDPGT. The potential of Ad5 vectors for delivering hepatic gene therapy in animal models is well-established, but relatively few studies have explored the utility of administering these vectors in the neonatal setting. The current study was undertaken to examine the efficacy of neonatal gene therapy in the Gunn rat model of CN type 1, and to determine whether the phenomenon of immune tolerance might allow for repeat administration later in life. Ad5-mediated gene therapy effectively complemented the bilirubin-UDPGT deficiency, resulting in substantial reductions in plasma bilirubin over a 3-month period. Furthermore, a second administration of gene therapy at 3 months of age was found to be similarly effective in reducing plasma bilirubin levels. The efficacy of Ad5-mediated gene therapy in neonates suggests that this approach might be effective against other hepatic disorders including autosomal recessive deficiencies in lipid metabolism and vascular homeostasis.