Crigler-Najjar Research Articles

Transient Increase in Intrahepatic Pressure Mediates Successful Treatment of the Gunn Rat with Reduced Doses of Lentiviral Vector

Lentiviral vectors can stably transduce hepatocytes and are promising tools for gene therapy of hepatic diseases. Although hepatocytes are accessible to blood-borne viral vectors through fenestrations of the hepatic endothelium, improved liver transduction after delivery of vectors to the blood stream is needed. As the normal endothelial fenestration and lentiviral vectors are similar in size (150 nm), we hypothesized that a transient increase in hepatic blood pressure may enhance in vivo gene transfer to hepatocytes. We designed a simple surgical procedure, by which the liver is temporarily excluded from blood flow. Lentiviral vectors were injected in a large volume to increase intrahepatic pressure. We demonstrated that in the Gunn rat, a model of Crigler-Najjar disease, the administration of low vector doses (corresponding to a multiplicity of infection of 0.2) by this procedure resulted in therapeutic correction of hyperbilirubinemia, without toxicity. The correction was sustained for 10 months (end of study). The same vector amounts yielded only partial correction after intraportal delivery. We believe that this new and clinically applicable strategy may broaden the range of genetic liver diseases accessible to gene therapy.

Hepatocyte Transplantation (HT) after Conformal Liver Irradiation (LIR) Enables Regiospecific Repopulation of Donor Cells with Amelioration of Hyperbilirubinemia in the Gunn Rat Model of Crigler-Najjar Syndrome

Hepatocyte Transplantation (HT) after Conformal Liver Irradiation (LIR) Enables Regiospecific Repopulation of Donor Cells with Amelioration of Hyperbilirubinemia in the Gunn Rat Model of Crigler-Najjar Syndrome

Generation of Liver Disease-Specific Induced Pluripotent Stem Cells Along with Efficient Differentiation to Functional Hepatocyte-Like Cells

The availability of disease-specific induced pluripotent stem cells (iPSCs) offers a unique opportunity for studying and modeling the effects of specific gene defects on human liver development in vitro and for testing small molecules or other potential therapies for relevant liver disorders. Here we report, for the first time, the derivation of iPSCs by the retroviral transduction of Yamanaka's factors in serum and feeder-free culture conditions from liver-specific patients with tyrosinemia, glycogen storage disease, progressive familial hereditary cholestasis, and two siblings with Crigler-Najjar syndrome. Furthermore, they were differentiated into functional hepatocyte-like cells efficiently. These iPSCs possessed properties of human embryonic stem cells (hESCs) and were successfully differentiated into three lineages that resembled hESC morphology, passaging, surface and pluripotency markers, normal karyotype, DNA methylation, and differentiation. The hepatic lineage-directed differentiation showed that the iPSC-derived hepatic cells expressed hepatocyte-specific markers. Their functionality was confirmed by glycogen and lipid storage activity, secretion of albumin, alpha-fetoprotein, and urea, CYP450 metabolic activity, as well as LDL and indocyanin green uptake. Our results provide proof of principal that human liver-disease specific iPSCs present an exciting potential venue toward cell-based therapeutics, drug metabolism, human liver development and disease models for liver failure disorders.

OP05 Medium term outcome of de novo autoimmune hepatitis after liver transplantation in children; a single centre experience

IntroductionPost-transplant de novo autoimmune hepatitis (dn-AIH) is a cause of late graft dysfunction characterized by hypergammaglobulinemia, elevated titres of serum auto-antibodies, histological features of chronic hepatitis with portal and periportal...

Monitoring of Intraportal Liver Cell Application in Children

Despite recent advances and promising results in children, liver cell transplantation (LCT) should still be regarded as an experimental therapy. Several substantial complications are known from animal studies and individual patients. However, safety data on liver cell infusion in children are scarce. We used LCT in four children of different ages (3 weeks to 11 years, 3-40 kg) and underlying diseases [acute liver failure (n = 1), urea cycle disorders (n = 2), and Crigler-Najjar syndrome (n = 1)]. Vital parameters, portal vein flow (PVF), portal vein pressure (PVP), and liver enzymes were measured every 5 min during cell application and hourly thereafter between applications. An application protocol with discontinuation rules depending on changes in PVF and PVP was developed and successfully applied. Application was feasible in all children despite the catastrophic overall condition of the patient with acute liver failure. No application-related changes in vital parameters were found, and none of the children experienced clinical signs of portal vein thrombosis, pulmonary embolism, or anaphylactic reactions. Time courses for changes in PVF, PVP, and liver enzymes were obtained. Thorough monitoring of portal vein pressure and duplex sonography according to a defined protocol is likely to increase safety of cell application in pediatric LCT.

388. Direct Injection of HDAd into the Liver Is as Effective but Less Toxic Than Intravenous Injection for Crigler-Najjar Syndrome Gene Therapy

388. Direct Injection of HDAd into the Liver Is as Effective but Less Toxic Than Intravenous Injection for Crigler-Najjar Syndrome Gene Therapy

Crigler Najjar syndrome type 2

Background: Crigler Najjar syndrome (CNS) is an extremely rare disorder of bilirubin metabolism that follows an autosomal recessive pattern of inheritance. CNS is elicited by a lack or deficiency of the enzyme uridine diphosphate glucuronyl transferase (UDGT). Two distinct forms have been described type 1 and type 2. Type 1 CNS is associated with neonatal unconjugated hyperbilirubinemia and kernicterus, which results in very high levels of unconjugated hyperbilirubinemia (up to 50 mg/dL) at birth. Type 2 CNS presents with serum bilirubin levels up to 20 mg/dL and markedly depressed activity of hepatic UDGT are characteristic of type 2 CNS. Objective: To rule out other causes for unconjugated hyperbilirubinemia, and arrive at a definitive diagnosis of Crigler Najjar syndrome type 2. Methods: Clinical examination of the baby and laboratory investigations like hemoglobin, complete blood count, thyroid profile, liver function tests and ultrasound were carried out. Results: Except for unconjugated hyperbilirubinemia, all other reports were within the normal range. Conclusion: Laboratory investigations and the response of the baby to phototherapy and phenobarbitone with decrease in the serum unconjugated bilirubin levels lead to a diagnosis of Crigler Najjar syndrome type 2. Hepatic glucuronyl transferase activity in a liver specimen, obtained by a closed biopsy and in vitro expression of mutant DNA from patient fibroblast, would have been helpful for definitive diagnosis.

Functional Characterization of Hepatocytes for Cell Transplantation: Customized Cell Preparation for Each Receptor

The first indication of hepatocyte transplantation is inborn liver-based metabolic disorders. Among these, urea cycle disorders leading to the impairment to detoxify ammonia and Crigler-Najjar Syndrome type I, a deficiency in the hepatic UDP-glucuronosyltransferase 1A1 present the highest incidence. Metabolically qualified human hepatocytes are required for clinical infusion. We proposed fast and sensitive procedures to determine their suitability for transplantation. For this purpose, viability, attachment efficiency, and metabolic functionality (ureogenic capability, cytochrome P450, and phase II activities) are assayed prior to clinical cell infusion to determine the quality of hepatocytes. Moreover, the evaluation of urea synthesis from ammonia and UDP-glucuronosyltransferase 1A1 activity, a newly developed assay using beta-estradiol as substrate, allows the possibility of customizing cell preparation for receptors with urea cycle disorders or Crigler-Najjar Syndrome type I. Sources of human liver and factors derived from the procurement of the liver sample (warm and cold ischemia) have also been investigated. The results show that grafts with a cold ischemia time exceeding 15 h and steatosis should not be accepted for hepatocyte transplantation. Finally, livers from non-heart-beating donors are apparently a potential suitable source of hepatocytes, which could enlarge the liver donor pool.

Persistent jaundice in an infant with homozygous beta thalassemia due to co-inherited Crigler-Najjar syndrome

Clinically apparent jaundice is unusual in patients with beta-thalassemia major. Co-inheritance of Gilbert syndrome has been reported to cause hyperbilirubinemia in these subjects. Crigler-Najjar syndrome is another rare disorder of bilirubin metabolism caused by mutation in the gene coding the enzyme UGT1A1. We report a patient of beta-thalassemia major who presented with persistent jaundice due to co-inherited Crigler-Najjar syndrome type 2 secondary to a novel mutation in UGT1A1 gene [homozygous base substitution at position 362 (GGT>AGT) in exon 3].

Liver cell transplantation in children

With increasing shortage of donor organs and changes in organ allocation systems, there is a considerable need for alternative or supportive techniques in the field of liver transplantation. Liver cell transplantation (LCT) is such an innovative technique that is especially promising for use in children, because it is less invasive than orthotopic liver transplantation (OLT). Two main indications exist: acute liver failure (ALF) and hepatic-based inborn errors of metabolism. Ten children with ALF underwent LCT so far. One patient recovered without OLT, five were successfully bridged to OLT, and four died. Clinical improvement like reduced hyperammonemia, improved coagulation, and regression of hepatic encephalopathy occurred in the majority of patients. For the treatment of inborn errors of metabolism, experience exists with Crigler-Najjar syndrome type 1 (CNS 1, n = 5) and urea cycle disorders (UCD, n = 6). All but two patients experienced metabolic stabilization for a mean period of 9.7 months, before the majority of children underwent OLT. Whereas transplant success in CNS 1 patients can be easily monitored by plasma bilirubin levels, it is much more difficult in UCDs, where many factors can contribute to clinical deterioration. Nevertheless, these individual therapeutic attempts of LCT yielded encouraging results, and prospective studies should be initiated.

Crigler-Najjar syndrome in The Netherlands: Identification of four novelUGT1A1alleles, genotype–phenotype correlation, and functional analysis of 10 missense mutants

Crigler-Najjar syndrome (CN), caused by deficiency of UGT isoform 1A1 (UGT1A1), is characterized by severe unconjugated hyperbilirubinemia. In this study we have analyzed 19 CN patients diagnosed in The Netherlands (18) and in Belgium (1), and have identified 14 different UGT1A1 mutations, four of which are novel. Two mutations were present in several unrelated patients, suggesting the presence of two founder effects in The Netherlands. In addition, we show linkage of the UGT1A1 *28 promoter polymorphism (rs5719145insTA) to three structural mutations. Functional studies of partial active UGT1A1 mutants are limited. Therefore, we performed in vitro studies to determine the functional activity of seven missense mutants identified in this study and of three reported previously. In addition to bilirubin, we also determined their activity toward eight other UGT1A1 substrates. We demonstrate that five mutants have residual activity that, depending on the substrate, varies from not detectable to 94% of wild-type UGT1A1 activity. The identification of four novel pathogenic mutations and the analysis of residual activity of 10 UGT1A1 missense mutants are useful for clinical diagnosis, and provides new insights in enzyme activity, whereas the identification of two founder mutations will speed up genetic counseling for newly identified CN patients in The Netherlands.

Total knee arthroplasty and Crigler–Najjar syndrome: A case report

Crigler–Najjar (CN) syndrome is a rare genetic disease characterized by hyperbilirubinemia due to a deficiency in the hepatic enzyme UDP-glucuronosyl-transferase. We describe the first case of total knee arthroplasty in a patient with CN syndrome (type II). This procedure was complicated by kernicterus 1 week after hospital discharge. He also developed Klebsiella bacteremia and sepsis, requiring a brief ICU stay. He was discharged in good condition 2 months later. It is evident that physicians involved in the care of patients with CN syndrome in the peri-operative period need to have a high index of suspicion for the development of severe hyperbilirubinemia and kernicterus in order to appropriately manage and, possibly, prevent this complication. A literature review and intra-operative observations provide insight into the possible relationship between hyperbilirubinemia and osteoarthritis as well as the peri-operative considerations to be made for this group of patients.

Living Related Liver Transplantation in Crigler-Najjar Syndrome Type 1

Four children underwent living related liver transplantation because of Crigler-Najjar syndrome type 1. Three were infants aged 2, 8(1/2), and 15 months, and weighed 5, 8, and 10 kg, respectively. Pretransplantation unconjugated bilirubin concentration was 22 to 30 mg/dL despite 12 to 14 hours of phototherapy daily. Patient 1, the 2-month-old infant, with unconjugated bilirubin concentration of 30 mg/dL, had a high-pitched cry, suggestive of bilirubin encephalopathy; results of neurologic examination were normal. Plasmapheresis and urgent liver transplantation were performed. Patient 4, a 13-year-old girl, had learning difficulties at school and attended a special class. Three patients received left lateral liver segments, and 1 patient received a left lobe. Biliary reconstruction was completed with duct-to-duct anastomosis. Bile leakage developed at the anastomosis in 2 patients, which was treated successfully with cholangioplasty. In all patients, the unconjugated bilirubin concentration normalized by day 1 posttransplantation, and no phototherapy was necessary. After transplantation, the 2-month-old infant with suspected encephalopathy exhibited hypotonia, spasticity of the lower extremities, and lack of head control. He died after vomitus aspiration during sleep at 10 months posttransplantation. The other 3 patients are alive with normal neurodevelopmental milestones. Irreversible brain damage may occur early in the course of Crigler-Najjar syndrome type 1. Urgent treatment including plasmapheresis, exchange transfusion, phototherapy, and liver transplantation may not reverse brain damage. Young infants must be evaluated carefully for subtle signs and symptoms of bilirubin encephalopathy. Liver transplantation is curative if performed before development of neurologic dysfunction.

Prediction of Deleterious Non-synonymous Single-Nucleotide Polymorphisms of Human Uridine Diphosphate Glucuronosyltransferase Genes

UDP glucuronosyltransferases (UGTs) are an important class of Phase II enzymes involved in the metabolism and detoxification of numerous xenobiotics including therapeutic drugs and endogenous compounds (e.g. bilirubin). To date, there are 21 human UGT genes identified, and most of them contain single-nucleotide polymorphisms (SNPs). Non-synonymous SNPs (nsSNPs) of the human UGT genes may cause absent or reduced enzyme activity and polymorphisms of UGT have been found to be closely related to altered drug clearance and/or drug response, hyperbilirubinemia, Gilbert's syndrome, and Crigler-Najjar syndrome. However, it is unlikely to study the functional impact of all identified nsSNPs in humans using laboratory approach due to its giant number. We have investigated the potential for bioinformatics approach for the prediction of phenotype based on known nsSNPs. We have identified a total of 248 nsSNPs from human UGT genes. The two algorithms tools, sorting intolerant from tolerant (SIFT) and polymorphism phenotyping (PolyPhen), were used to predict the impact of these nsSNPs on protein function. SIFT classified 35.5% of the UGT nsSNPs as "deleterious"; while PolyPhen identified 46.0% of the UGT nsSNPs as "potentially damaging" and "damaging". The results from the two algorithms were highly associated. Among 63 functionally characterized nsSNPs in the UGTs, 24 showed altered enzyme expression/activities and 45 were associated with disease susceptibility. SIFT and Polyphen had a correct prediction rate of 57.1% and 66.7%, respectively. These findings demonstrate the potential use of bioinformatics techniques to predict genotype-phenotype relationships which may constitute the basis for future functional studies.

Long-term reduction of jaundice in Gunn rats by nonviral liver-targeted delivery of Sleeping Beauty transposon #

Asialoglycoprotein receptor (ASGPR)-mediated endocytosis has been used to target genes to hepatocytes in vivo. However, the level and duration of transgene expression have been low because of lysosomal translocation and degradation of the DNA and lack of its integration into the host genome. In this study we packaged the DNA of interest in proteoliposomes containing the fusogenic galactose-terminated F-glycoprotein of the Sendai virus (FPL) for targeted delivery to hepatocytes. After the FPL binds to ASGPR on the hepatocyte surface, fusogenic activity of the F-protein delivers the DNA into the cytosol, bypassing the endosomal pathway. For transgene integration we designed plasmids containing one transcription unit expressing the Sleeping Beauty transposase (SB) and another expressing human uridinediphosphoglucuronate glucuronosyltransferase-1A1 (pSB-hUGT1A1). The latter was flanked by inverted/direct repeats that are substrates of SB. In cell culture, FPL-mediated delivery of the E. coli beta-galactosidase gene (LacZ) resulted in transduction of ASGPR-positive cells (rat hepatocytes or Hepa1 cell line), but not of ASGPR-negative 293 cells. Intravenous injection of the FPL-entrapped pSB-hUGT1A1 (4-8 microg/day, 1-4 doses) into UGT1A1-deficient hyperbilirubinemic Gunn rats (model of Crigler-Najjar syndrome type 1) resulted in hUGT1A1 expression in 5%-10% of hepatocytes, but not in other cell types. Serum bilirubin levels declined by 30% +/- 4% in 2 weeks and remained at that level throughout the 7-month study duration. With histidine containing FPL, serum bilirubin was reduced by 40% +/- 5%, and bilirubin glucuronides were excreted into bile. No antibodies were detectable in the recipient rats against the F-protein or human UGT1A1. FPL is an efficient hepatocyte-targeted gene delivery platform in vivo that warrants further exploration toward clinical application.

A Step Toward Liver Gene Therapy: Efficient Correction of the Genetic Defect of Hepatocytes Isolated From a Patient With Crigler-Najjar Syndrome Type 1 With Lentiviral Vectors

Ex vivo liver gene therapy may be a future alternative to orthotopic liver transplantation for the treatment of some liver diseases. We previously described the transduction in suspension with lentiviral vectors and immediate hepatocyte transplantation (SLIT) protocol and its high transduction rate with normal human hepatocytes. We also reported SLIT efficiency in the animal model of Crigler-Najjar type 1 syndrome (CN-1), the Gunn rat. Here, we evaluated SLIT efficiency with diseased human hepatocytes. Hepatocytes of the liver from a 4-year-old patient presenting CN-1 were isolated. They were transduced with liver-specific lentiviral vectors expressing uridine-diphosphate-glucuronosyltransferase (hUGT1A1) or green fluorescent protein, and then analyzed in vitro for transduction efficiency and hUGT1A1 expression, or transplanted in nonobese diabetic/severe combined immunodeficiency (SCID) mice to evaluate long-term survival of transplanted cells. More than 90% of CN-1 hepatocytes were transduced. Hepatocytes produced hUGT1A1 protein after lentiviral transduction. After having been subjected to the SLIT, lentivirally transduced CN-1 hepatocytes engrafted long term (up to 26 weeks posttransplantation) in recipient livers and expressed green fluorescent protein or hUGT1A1 vector. The SLIT protocol allowed for a high transduction of CN-1 hepatocytes and restoration of the expression of the deficient protein. Furthermore, long-term survival of lentivirally transduced CN-1 hepatocytes in the liver of immunodeficient mice was demonstrated. This study is therefore an important step toward human application of lentiviral gene therapy.

Towards Liver-Directed Gene Therapy for Crigler-Najjar Syndrome

Crigler-Najjar (CN) syndrome is a recessive inherited disorder caused by deficiency of uridine diphospho-glucuronosyl transferase 1A1. This hepatic enzyme catalyzes the glucuronidation of bilirubin, an essential step in excretion into bile of this neurotoxic compound. As a result, CN patients suffer from severe unconjugated hyperbilirubinemia and are at risk of bilirubin encephalopathy. Over the last decades ex vivo and in vivo gene therapy using viral and non-viral vectors has been used to correct hyperbilirubinemia in the relevant animal model for CN syndrome, the Gunn rat. Several of these approaches did result in long-term correction of serum bilirubin levels in this animal model. However, none have been translated into a clinical trial. In this review we will recapitulate the strategies used and discuss their suitability for clinical application in the near future. We will also address specific safety measures in the gene therapy protocol needed to prevent adverse effects such as bilirubin toxicity. Since CN seems an ideal model for other monogenetic inherited metabolic liver disorders, development of liver-directed gene-therapy has relevance beyond this rare disease.

Characterization of in vitro and in vivo metabolism of AG-024322, a novel cyclin-dependent kinase (CDK)

Uncontrolled cell proliferation is the hall mark of many cancers, and is typically manifested by a deregulation of the cell-division cycle. CDKs play critical roles in regulating cell cycle, apop- tosis and cell differentiation. AG-024322 is a multitargeted CDK inhibitor that has been shown to induce cancer cell apoptosis and de- monstrate significant antitumor activity in hu-man tumor xenograft models. This compound is under clinical development as an intravenous anticancer agent. AG-024322 exhibited moder-ate to high systemic clearance across preclini-cal species. In vitro metabolism in human liver microsomes and hepatocytes demonstrates that glucuronidation and oxidation represent the major metabolic pathways of AG-024322. The experiments of chemical inhibition and micro-somes containing individual CYP or UGT iso-forms revealed that CYP3A and UGT1A1 appear to predominantly mediate AG-024322 oxidation and glucuronidation, respectively. Formation kinetics of the two pathways in human liver mi-crosomes suggested that the glucuronidation activity of AG-024322 was approximately 3-fold higher as compared to CYP-mediated oxidation, contributing approximately 37% and 13% of the total clearance, respectively, based on the pro-jected human clearance. UGT1A1 is a poly-morphic isoform involved in glucuronidation of bilirubin. It is of concern if glucuronidation via UGT1A1 plays a major role in the elimination of AG-024322 in humans as competitive inhibition of UGT1A1 has been associated with toxicity (Gilbert and Crigler-Najjar syndromes). There-fore, this information was used to influence the clinical study design to only include subjects having constitutive expression of UGT1A1 in the first human study, thereby decreasing the potential risk of toxicity to patients.

PGD with blastomere analysis for Crigler–Najjar syndrome; UGT1A1 analysis with aneuploidy testing

PGD with blastomere analysis for Crigler–Najjar syndrome; UGT1A1 analysis with aneuploidy testing

Review of hepatocyte transplantation

Hepatocyte transplantation is a promising treatment for several liver diseases and can also be used as a "bridge" to liver transplantation in cases of liver failure. Although the first animal experiments with this technique began in 1967, it was first applied in humans only in 1992. Unfortunately, unequivocal evidence of transplanted human hepatocyte function has been obtained in only one patient with Crigler-Najjar syndrome type I and, even then, the amount of bilirubin-UDP-glucuronosyltransferase enzyme activity derived from the transplanted cells was not sufficient to eliminate the patient's eventual need for organ transplantation. A literature review was carried out using MEDLINE and library searches. This review considers the following: (1) alternatives or bridges to orthotopic liver transplantation (OLT); (2) solutions to the shortage of organs-the shortage of organ donors has impeded the development of human hepatocyte transplantation, and immortalized hepatocytes in particular could provide an unlimited supply of transplantable cells in a nearly future; (3) future directions. We review these efforts along with hepatocyte transplantation over the last 13 years.