A chimeric RNA/DNA oligonucleotide was constructed to induce a sequence mutation in the rat factor IX gene, resulting in prolonged coagulation. Oligonucleotides were targeted to hepatocytes in cell culture or in vivo by intravenous injection. Nucleotide conversion was both site-specific and dose-dependent. The mutated gene was associated in vivo with significantly reduced factor IX coagulant activity and a marked prolongation of the activated partial thromboplastin time. The results demonstrate that single base-pair alterations can be introduced in hepatocytes in situ by RNA/DNA oligonucleotides, suggesting a potentially powerful strategy for hepatic gene repair without the use of viral vectors.

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 ...

This paper explores the dynamics of price discovery between the Dow Jones Industrial Average (DJIA) index and its three derivative products: the DIAMOND exchange-traded fund (ETF), the floor-traded regular futures, and the electronically traded mini futures. Even though the American Stock Exchange is the primary listing exchange for the ETF, the analysis indicates that the electronically traded ETF on the Archipelago (ArcaEx) electronic communications network dominates the price discovery process for DIAMOND shares. The E-mini futures contribute the most to price discovery, followed by the ArcaEx DIAMOND. The DJIA index and regular futures contribute least to price discovery. The analysis is repeated using the derivatives of the S&P 500 index as a robustness check. The results indicate that multi-market trading ensures greater pricing efficiency. Informed traders favor electronic trading because of immediate and anonymous trade execution. Copyright 2006 The Authors Journal compilation (c) 2006 Blackwell Publishing Ltd.