Lifelong elimination of hyperbilirubinemia in the Gunn rat with a single injection of helper-dependent adenoviral vector

Abstract

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 mg/dl to <<1.4 mg/dl for >2 yr after a single i.v. administration of vector expressing the therapeutic transgene at a dose of 3 x 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 IIIalpha and XIIIalpha 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 x 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.

Fig. 1.

HD-Ad administration into Gunn rats: effect on serum bilirubin levels. Eight-week-old female Gunn rats received saline solution or HD-Ad PEPCK UGT1A1 by tail vein injection. The dose of HD-Ad vector is expressed as vp per kg. (a) Photographs of 10-μl samples of rat serum from a representative animal from each group. (b) Serum total bilirubin levels by diazo determination. Data are indicated as mean ± SEM. The difference between the HD-Ad-treated and the saline control groups was statistically significant for all points analyzed. (c) HPLC analysis of serum from a WT rat, a high-dose rat (115 wk after treatment), an intermediate-dose rat (102 wk after treatment), and a low-dose rat (107 wk after treatment). Unconjugated bilirubin (red peak) concentrations based on HPLC analyses were 0.1, 0.1, 0.7, and 1.9 mg/dl, respectively. The top chromatogram shows, for comparison, a typical chromatogram of serum from a healthy human adult (0.4 mg/dl). Blue peaks are the injection front.

Fig. 2.

HD-Ad-treated Gunn rats: bilirubin glucuronides in bile. HPLC chromatograms of bile from a WT control rat (a), a high-dose rat (115 wk posttreatment) (b), an intermediate-dose rat (102 wk posttreatment) (c), and a low-dose rat (107 wk posttreatment) (d). The lower chromatograms are of bile from a saline-treated homozygous Gunn rat control. BDG, bilirubin diglucuronide; BMG, bilirubin monoglucuronide; UCB, unconjugated bilirubin. (Note that the vertical scale in d is greatly expanded compared with a–c.)

Fig. 3.

HD-Ad-treated Gunn rats: bilirubin isomer challenge. A mixture of bilirubins IIIα (≈0.25 mg) and XIIIα (≈0.25 mg) in 1 ml of rat serum was injected i.v. into a WT control, a high-dose rat (115 wk after vector administration), and an intermediate-dose rat (102 wk after vector administration). Bile was collected just before (t = 0) and 6–15 min after isomer injection and analyzed by HPLC. (a) Representative mixture of isomers injected. (b–d) HPLC of bile from the control, high-dose, and intermediate-dose rats, respectively. BR, bilirubin; DG, diglucuronide; MG, monoglucuronide.

Fig. 4.

HD-Ad administration into Gunn rats: mRNA and protein analysis. (a) RT-PCR amplification from total liver RNA extracted from Gunn rats 20 and 52 wk after administration of saline solution or 3 × 10¹² vp/kg HD-Ad. Ethidium bromide-stained agarose gel shows human UGT1A1 and rat β-globin amplified fragments. Lane 1, 1 kb of Plus DNA ladder (Invitrogen); lane 2, PCR with no DNA template control; lane 3, PCR using plasmid with human UGT1A1 cDNA as template; lane 4, PCR using HD-Ad PEPCK UGT1A1 DNA as template; lane 5, no reverse transcriptase control for lane 6; lane 6, RT-PCR using liver RNA from saline-treated Gunn rat 20 wk postinjection as template; lane 7, RT-PCR using liver RNA from HD-Ad-treated Gunn rat 20 wk postinjection as template; lane 8, no reverse transcriptase control for lane 7; lane 9, RT-PCR using liver RNA from saline-treated Gunn rat 52 wk postinjection as template; and lane 10, RT-PCR using liver RNA from HD-Ad-treated Gunn rat 52 wk postinjection as template. (b) Western blot analysis of human UGT1A1 expression in liver homogenates. Protein homogenates from liver biopsy of Gunn rats 20 weeks after administration of saline solution or 3 × 10¹² vp/kg HD-Ad PEPCK UGT1A1 were resolved by SDS/PAGE (7.5%). Immunoblot analysis was performed by using anti-human UGT1A1 mAbs. Lane 1, PBS-treated rat, 100 μg total protein; lane 2, HD-Ad-treated rat, 100 μg total protein; lane 3, PBS-treated rat, 25 μg microsomal protein; and lane 4, HD-Ad-treated rat, 25 μg microsomal protein. (c and d) Immunohistochemical analysis of human UGT1A1 expression in liver sections. Sections of liver biopsy of Gunn rats 20 wk after administration of saline solution (c) or 3 × 10¹² vp/kg HD-Ad (d) were immunostained by using specific anti-human UGT1A1 antibodies as primary antibody. Brown staining in d indicates UGT1A1.

Fig. 5.

HD-Ad administration into Gunn rats: toxicity profile. Serum alanine aminotransferase (ALT) levels (a) and circulating platelet counts (b) in Gunn rats 3 days and 3 weeks after tail vein administration of saline or HD-Ad. Doses of HD-Ad are expressed as vp per kg of body weight. Data are indicated as mean ± SEM. ND, not determined.