N Roy Chowdhury

A non-immunogenic adenoviral vector, coexpressing CTLA4Ig and bilirubin-uridine-diphosphoglucuronateglucuronosyltransferase permits long-term, repeatable transgene expression in the Gunn rat model of Crigler-Najjar syndrome.

Host immune responses limit the duration of expression of transgenes introduced by recombinant adenoviruses, preclude gene transfer upon vector readministration and cause liver injury. CTLA4Ig inhibits immune response by blocking the co-stimulatory interaction between CD28 on T cells and B7 on antigen-presenting cells. We have constructed a recombinant adenovirus, Ad-hUGT1A1-CTLA4Ig that coexpresses human bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (hUGT1A1) and soluble murine CTLA4Ig, both driven by CMV immediate–early promoters. After intravenous injection of this vector (6 × 1011 p.f.u.) into UGT1A1-deficient jaundiced Gunn rats, serum CTLA4Ig levels peaked at 1.8–2.0 mg/ml on day 7 and declined thereafter to 0.2 mg/ml by day 180. Serum bilirubin declined from mean preinjection levels of 8.0 mg/dl to 0.48–0.6 mg/dl in 3 days, remained normal for 28 weeks, and then gradually increased to 8 mg/dl by day 350. A second injection of Ad-hUGT1A1-CTLA4Ig normalized serum bilirubin. In two rats in this group that were followed longer, serum bilirubin increased to 3.1 and 3.5 mg/dl in 40 weeks, but was normalized again after a third injection. The antibody and cytotoxic lymphocyte (CTL) responses were negligible, and liver biopsy showed no inflammatory cell infiltration. Rats receiving a tertiary challenge with Ad-LacZ (expressing E. coli β-galactosidase) (5 × 1011 p.f.u.), 2 months after the second dose of Ad-hUGT1A1-CTLA4Ig, showed β-galactosidase expression in over 80% of hepatocytes. In contrast, after Ad-hUGT1A1 (which expresses UGT1A1 alone) injection, serum bilirubin remained normal for only 4 weeks, and returned to preinjection levels by day 120. Bilirubin levels did not decline upon reinjection, and β-galactosidase was not expressed after Ad-LacZ. High levels of adenovirus-specific antibodies and CTL, and hepatic inflammation were found. This is the first demonstration that coexpression of CTLA4Ig permits prolonged expression and repeatable gene transfer by an adenoviral vector.

Differential regulation by triiodothyronine of substrate-specific uridinediphosphoglucuronate glucuronosyl transferases in rat liver

Hepatic uridinediphosphoglucuronate glucuronosyl transferase (UDPglucuronyltransferase, EC 2.4.1.17) is functionally heterogeneous; 4-nitrophenol and bilirubin are representative substrates for two functional forms of the enzyme. UDPglucuronyltransferase activity for bilirubin and 4-nitrophenol was separated from solubilized rat liver microsomes by DEAE-cellulose chromatography and corresponding enzymes were purified. A radioimmunoassay was developed using a rabbit antiserum against purified rat 4-nitrophenol-specific UDPglucuronyltransferase, which precipitated enzyme activities toward both 4-nitrophenol and bilirubin. After treatment with triiodothyronine (T3) (0.55 mg/kg body weight), hepatic microsomal UDPglucuronyltransferase activity for 4-nitrophenol was increased 400% as compared to controls; the enzyme activity for bilirubin was decreased by 80%; the changes in the substrate-specific enzyme activities were reflected in the enzymatically active fractions separated after DEAE-cellulose chromatography. The changes in enzyme activities paralleled changes in the concentrations of the two corresponding UDP glucuronyltransferase proteins in the chromatographic fractions, as measured by radioimmunoassay. The results indicate that the opposite effects of T3 on the two forms of UDPglucuronyltransferase activity is due to its differential effect on corresponding enzyme proteins.

Bilirubin Diglucuronide Formation in Intact Rats and in Isolated Gunn Rat Liver

Bilirubin diglucuronide (BDG) may be formed in vitro by microsomal UDP glucuronosyl transferase (EC 2.4.1.17)-mediated transfer of a second mole of glucuronic acid from UDP-glucuronic acid, or by dismutation of bilirubin monoglucuronide (BMG) to BDG and unconjugated bilirubin, catalyzed by an enzyme (EC 2.4.1.95) that is concentrated in plasma membrane-enriched fractions of rat liver. To evaluate the role of these two enzymatic mechanisms in vivo, [(3)H]bilirubin mono-[(14)C]glucuronide was biosynthesized, purified by thin-layer chromatography, and tracer doses were infused intravenously in homozygous Gunn (UDP glucuronyl transferase-deficient) rats or Wistar rats. Bilirubin conjugates in bile were separated by high-pressure liquid chromatography and (3)H and (14)C were quantitated. In Gunn rats, the (14)C:(3)H ratio in BDG excreted in bile was twice the ratio in injected BMG. In Wistar rats the (14)C:(3)H ratio in biliary BDG was 1.25 +/- 0.06 (mean +/- SEM) times the ratio in injected BMG. When double labeled BMG was injected in Wistar rats after injection of excess unlabeled unconjugated bilirubin (1.7 mumol), the (14)C:(3)H ratio in BDG excreted in bile was identical to the ratio in injected BMG. Analysis of isomeric composition of bilirubin conjugates after alkaline hydrolysis or alkaline methanolysis indicated that the bile pigments retained the IX(alpha) configuration during these experiments. The results indicate that both enzymatic dismutation and UDP glucuronyl transferase function in vivo in BDG formation, and that dismutation is inhibited by a high intrahepatic concentration of unconjugated bilirubin. This hypothesis was supported by infusion of [(3)H]bilirubin-monoglucuronide in isolated perfused homozygous Gunn rat liver after depletion of intrahepatic bilirubin by perfusion with bovine serum albumin (2.5%), and after bilirubin repletion following perfusion with 0.34 mM bilirubin. From 20 to 25% of injected radioactivity was recovered in BDG in bile in the bilirubin-depleted state; only 8-10% of radioactivity was in BDG in bile after bilirubin repletion. After infusion of [(3)H]bilirubin di-[(14)C]glucuronide in homozygous Gunn rats, 5-7% of the injected pigment was excreted in bile as BMG. The (14)C:(3)H ratio in the injected BDG was 10% greater than the (14)C:(3)H ratio in BMG excreted in bile. These results indicate that in vivo, dismutation rather than partial hydrolysis, is responsible for BMG formation. Incubation of [(3)H]bilirubin, BDG and a rat liver plasma membrane preparation resulted in formation of BMG (3.3 nmol/min per mg protein) indicating that dismutation is also reversible in vitro.

Modification of hepatic genomic DNA using RNA/DNA oligonucleotides

The ideal gene therapy is one that repairs the precise genetic defect without additional modification of the genome. Such a strategy has been developed for correcting single nucleotide mutations by using RNA/DNA oligonucleotides, or chimeraplasts. This approach for in situ repair is based on the delivery of exogenous DNA designed to mediate genomic base conversion, insertion, or deletion, thereby, correcting the genetic mutation. Using in vivo delivery systems to hepatocytes via the asialoglycoprotein receptor, we targeted rat liver DNA and successfully modified the genomic sequence by chimeraplasty. The changes in both the hepatic genes, and their associated phenotypes remained stable for 2 years. In addition, we also examined the potential to alter sequence defects in mitochondrial DNA. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeraplast-mediated DNA changes. Using an in vitro DNA repair assay of mutagenized plasmids and an Escherichia coli readout system, we showed that extracts from highly purified rat liver mitochondria have the essential enzymatic activity to mediate precise single-nucleotide changes at a frequency similar to liver nuclear extracts. Moreover, single-stranded oligonucleotides carrying a single nucleotide mismatch with the target sequence were capable of promoting gene conversion using either mitochondrial or nuclear extracts. Several approaches now exist for the precise repair of genetic mutations using either single-stranded or RNA/DNA chimeric oligonucleotides.

Use of Microbeads for Cell Transplantation

Transplantation of isolated hepatocytes and other animal cells have many potential therapeutic and investigative applications. Liver cell transplantation, if successful in man, could be used in managing acute liver failure, which at this time carries dismal prognosis. It could also possibly be used for replacement of specific deficient liver functions in inherited liver diseases. Finally, hepatocytes or other transplanted cells may have potential application as vehicles for somatic gene therapy.

Bile pigment composition and hepatic UDP-glucuronyl transferase activity in the fetal and adult dogfish shark, Squalus acanthias

Abstract 1. 1. Bile pigments from adult and fetal spiny dogfish (Squalus acanthias) were separated and quantitated by high pressure liquid chromatography. 2. 2. In order of relative abundance, bilirubin monoglucuronide, unconjugated bilirubin, bilirubin diglucuronide and bilirubin monoglucoside were present in adult bile and in fetal bile and meconium. 3. 3. All bile pigments were of IXα configuration. 4. 4. Specific hepatic microsomal UDP-glucuronate glucuronosyl transferase activity towards bilirubin and p- nitrophenol were comparable in fetal and adult dogfish.

Development of an efficient nonviral gene transfer method to hepatocytes using cationic liposome and ursodeoxycholic acid

• L0625 DEVELOPMENT OF AN EFFICIENT NONVIRAL GENE TRANSFER METHOD TO HEPATOCYTES USING CATIONIC LIPOSOME AND URSODEOXYCHOLIC ACID. K Tada, H Minami, Y Oka, H Iuchi, SMF Akbar, N Horiike, N Roy Chowdhury*, J Roy Chowdhury*, M Onji. The Third Dept of Internal Medicine, Ehime University School of Medicine, Japan, and the Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York, NY.