Retrovirus-mediated expression of HUG Br1 in Crigler-Najjar syndrome type I human fibroblasts and correction of the genetic defect in Gunn rat hepatocytes.

Crigler-Najjar syndrome type I (CN-I) is a congenital hepatic metabolic deficiency in bilirubin UDP-glucuronosyltransferase activity which leads to profound jaundice and death from kernicterus. UGT1, the gene locus coding for multiple glucuronosyltransferase isoforms, has been well characterized and the cDNA for the most active form, HUG Br1, has been cloned. Recent advances in liver directed gene transfer suggest that this disease could be treated through gene therapy. As an initial step to correct the genetic defect in Crigler-Najjar type I, recombinant retroviruses were used to transduce an HUG Br1 gene into hepatocytes of a rat model of CN-I and CN-I fibroblasts. The retroviral vector gagCMVBA HUG Br1 was constructed and helper-free amphotrophic virus was isolated and used to transfer bilirubin UDP-glucuronosyltransferase activity to genetically deficient cells. The efficiency of transduction as measured by Southern blot analysis of integrated proviral sequences in DNA of recipient cells ranged from 5 to 100%. HUG Br1 gene expression was documented by blot hybridization analysis of total cellular RNA, immunotransblot analysis using a rabbit polyclonal antipeptide HUG Br1 antibody, and lysate enzymatic assay of bilirubin UDP-glucuronosyltransferase activity. HUG Br1 gene transfer was definitively demonstrated by four independent modalities following HUG Br1 retroviral transduction.