Correction of the UDP-glucuronosyltransferase gene defect in the Gunn rat model of Crigler–Najjar syndrome type I with a chimeric oligonucleotide

Abstract

Crigler-Najjar syndrome type I is characterized by unconjugated hyperbilirubinemia resulting from an autosomal recessive inherited deficiency of hepatic UDPglucuronosyltransferase (UGT) 1A1 activity. The enzyme is essential for glucuronidation and biliary excretion of bilirubin, and its absence can be fatal. The Gunn rat is an excellent animal model of this disease, exhibiting a single guanosine (G) base deletion within the UGT1A1 gene. The defect results in a frameshift and a premature stop codon, absence of enzyme activity, and hyperbilirubinemia. Here, we show permanent correction of the UGT1A1 genetic defect in Gunn rat liver with site-specific replacement of the absent G residue at nucleotide 1206 by using an RNA͞DNA oligonucleotide designed to promote endogenous repair of genomic DNA. The chimeric oligonucleotide was either complexed with polyethylenimine or encapsulated in anionic liposomes, administered i.v., and targeted to the hepatocyte via the asialoglycoprotein receptor. G insertion was determined by PCR amplification, colony lift hybridizations, restriction endonuclease digestion, and DNA sequencing, and confirmed by genomic Southern blot analysis. DNA repair was specific, efficient, stable throughout the 6-month observation period, and associated with reduction of serum bilirubin levels. Our results indicate that correction of the UGT1A1 genetic lesion in the Gunn rat restores enzyme expression and bilirubin conjugating activity, with consequent improvement in the metabolic abnormality.UDP-glucuronosyltransferases (UGTs) are a family of membrane-bound enzymes that catalyze the conjugation of numerous xenobiotics and endogenous substrates with glucuronic acid. Of the known isoforms, only UGT1A1 is physiologically relevant in bilirubin glucuronidation and biliary excretion of this potentially toxic metabolite (1, 2). Crigler-Najjar (CN) syndrome is the inherited deficiency of hepatic UGT1A1 activity and is characterized by elevated serum levels of unconjugated bilirubin (3). Of the two types of CN syndrome, type I is more severe and is characterized by a nearly complete absence of UGT1A1 activity, whereas incomplete deficiency of the enzyme is associated with the less severe type II form (4, 5).The homozygous Gunn rat, a mutant strain of Wistar rat, is an accurate animal model for CN syndrome type I. Its liver lacks UGT1A1 activity because of the deletion of a single guanosine (G) base in UGT1A1 that results in a frameshift and a premature stop codon (6, 7). Recombinant adenoviral vectors have been used in vivo to correct the hyperbilirubinemia in the Gunn rat with persistent expression of the human bilirubin UGT1A1 enzyme for as long as 2 months (8, 9). Significant progress also has been made in overcoming the immunogenicity of the adenoviral-based vectors (9-11), but their use requires repeated treatments and immunomodulation of the host to maintain therapeutic levels of UGT1A1.A novel approach, based on mechanisms of DNA repair (12), was reported to correct single nucleotide mutations in ...

A comparison of gene repair strategies in cell culture using a lacZ reporter system

“…4 More recently, somatic cell gene targeting has been achieved in vitro with RNA:DNA chimeric oligonucleotides [5][6][7][8] double-and single-stranded PCR-generated fragments 2,3,9 and single-stranded oligonucleotides. Gene repair has also been reported in vivo in a variety of tissues including the liver, 10 , lung, 11 muscle, 12 and skin, 13 using a variety of fragments or oligonucleotides.…”

A LacZ-based transgenic mouse for detection of somatic gene repair events in vivo

“…Groups advocating RDOs as a strategy for gene repair believe that this may be due to differences in the cell type, target locus or quality control in oligonucleotide synthesis. It is difficult to find a supplier for RDOs; companies that provided material for some of the successful studies 8 are no longer providing this service.…”

“…The feasibility of somatic cell gene targeting has been demonstrated using RNA:DNA chimaeric oligonucleotides (RDOs) or single-stranded DNA oligonucleotides (ODN) in vitro [1][2][3][4][5][6] and in vivo. [6][7][8][9][10] Gene repair has also been reported in vivo, using short DNA fragments in the lung 11 and muscle. 9,12 It is difficult to compare the efficiency of in vivo gene repair methods used by different groups, because each experimental approach is very different.…”

“…9,12 It is difficult to compare the efficiency of in vivo gene repair methods used by different groups, because each experimental approach is very different. Methods of delivery can vary, for example, intravenous 8 and intraperitoneal 13 approaches have both been reported to target the liver. There are also variations in the loci of genes targeted by these approaches and in their levels of transcription, both of which may impinge upon the success of gene repair.…”

“…There are also variations in the loci of genes targeted by these approaches and in their levels of transcription, both of which may impinge upon the success of gene repair. 8 A mouse model bearing a mutated reporter gene such as LacZ allows sensitive detection of somatic gene repair and identification of cell types successfully targeted. A suitable locus for expression of such a reporter would be the Rosa26 locus, which directs ubiquitous expression in the embryo and the adult mouse.…”