• Congenital
• Egypt
• Liver diseases
• Orphan
• Rare

• Humans
• Egypt/epidemiology
• Liver Diseases/epidemiology
• Liver Diseases/diagnosis
• Rare Diseases/epidemiology
• Rare Diseases/diagnosis

• Crigler–Najjar syndrome type 2
• UGT1A1 deficiency
• clinical isolated jaundice
• indirect hyperbilirubinemia
• phenobarbitone
• unconjugated hyperbilirubinemia

• Croatia
• UGT1A1
• bilirubin UDP-glucuronosyltransferase
• case report
• case series
• crigler-Najjar syndrome
• hyperbilirubinemia

• Crigler-Najjar
• LNP
• UGT1A1
• hyperbilirubinemia
• lipid nanoparticle
• mRNA
• phototherapy

• UGT1A1
• encephalopathy
• liver transplantation
• phototherapy
• unconjugated hyperbilirubinemia

• Bilirubin
• Cost of Illness
• Crigler-Najjar Syndrome
• Glucuronosyltransferase
• Humans
• Registries

Liver transplantation (LT) remains the gold standard treatment for end stage liver disease in the pediatric population. For liver based metabolic disorders (LBMDs), the decision for LT is predicated on a different set of paradigms. With improved outcomes post-transplantation, LT is no longer merely life saving, but has the potential to also significantly improve quality of life. This review summarizes the clinical presentation, medical treatment and indications for LT for some of the common LBMDs. We also provide a practical update on the dilemmas and controversies surrounding the indications for transplantation, surgical considerations and prognosis and long terms outcomes for pediatric LT in LBMDs. Important progress has been made in understanding these diseases in recent years and with that we outline some of the new therapies that have emerged.

• Pediatric metabolic liver disease
• Liver transplantation
• Liver based metabolic disorders
• Inherited
• Cell therapy
• Gene therapy

• Crigler-Najjar syndrome
• diagnosis
• therapy

In contrast to AAV, Simian Virus 40 (rSV40) not inducing neutralizing antibodies (NAbs) allowing re-treatment seems a promising vector for neonatal treatment of inherited liver disorders. Several studies have reported efficacy of rSV40 in animal models for inherited liver diseases. In all studies the ubiquitous endogenous early promoter controlled transgene expression establishing expression in all transduced tissues. Restricting this expression to the target tissues reduces the risk of immune response to the therapeutic gene. In this study a liver specific rSV40 vector was generated by inserting a hepatocyte specific promoter. This increased the specificity of the expression of hUGT1A1 in vitro. However, in vivo the efficacy of rSV40 appeared too low to demonstrate tissue specificity while increasing the vector dose was not possible because of toxicity. In contrast to earlier studies, neutralizing antibodies were induced. Overall, the lack of a platform to produce high titered and pure rSV40 particles and the induction of NAbs, renders it a poor candidate for in vivo gene therapy.

• Animals
• Antibodies, Neutralizing/genetics
• Antibodies, Neutralizing/metabolism
• Cell Line, Tumor
• Genetic Therapy/methods
• Genetic Vectors/genetics
• Genetic Vectors/metabolism
• Glucuronosyltransferase/deficiency
• Glucuronosyltransferase/genetics
• Glucuronosyltransferase/metabolism
• Humans
• Hyperbilirubinemia, Hereditary/genetics
• Hyperbilirubinemia, Hereditary/pathology
• Liver/metabolism
• Mice
• Mice, Knockout
• Promoter Regions, Genetic
• Simian virus 40/genetics
• Tissue Distribution
• Transcriptional Activation

• China Scholarship Council (CSC)

A clinical trial using adeno-associated virus serotype 8 (AAV8)-human uridine diphosphate glucuronosyltransferase 1A1 (hUGT1A1) to treat inherited severe unconjugated hyperbilirubinemia (Crigler-Najjar syndrome) is ongoing, but preclinical data suggest that long-term efficacy in children is impaired due to loss of transgene expression upon hepatocyte proliferation in a growing liver. This study aims to determine at what age long-term efficacy can be obtained in the relevant animal model and whether immune modulation allows re-treatment using the same AAV vector. Neonatal, suckling, and juvenile Ugt1a1-deficient rats received a clinically relevant dose of AAV8-hUGT1A1, and serum bilirubin levels and anti-AAV8 neutralizing antibodies (NAbs) in serum were monitored. The possibility of preventing the immune response toward the vector was investigated using a rapamycin-based regimen with daily intraperitoneal (i.p.) injections starting 2 days before and ending 21 days after vector administration. In rats treated at postnatal day 1 (P1) or P14, the correction was (partially) lost after 12 weeks, whereas the correction was stable in rats injected at P28. Combining initial vector administration with the immune-suppressive regimen prevented induction of NAbs in female rats, allowing at least partially effective re-administration. Induction of NAbs upon re-injection could not be prevented, suggesting that this strategy will be ineffective in patients with low levels of preexisting anti-AAV NAbs.

• AAV
• Bilirubin
• Neutralizing Antibodies
• Rapamycin
• Unconjugated Hyperbilirubinemia

• Crigler—Najjar syndrome
• Fibrosis
• Liver histology

• Child
• Child, Preschool
• Crigler-Najjar Syndrome/genetics
• Cross-Sectional Studies
• Humans
• Infant
• Liver
• Liver Transplantation
• Retrospective Studies

• Bile
• Cholestasis
• Pruritogen
• Pruritus
• Receptor

• Animals
• Cholestasis/metabolism
• Cholestasis/pathology
• Humans
• Pruritus/metabolism
• Pruritus/pathology

Potency assessment of clinical-grade vector lots is crucial to support adeno-associated virus (AAV) vector release and is required for future marketing authorization. We have developed and validated a cell-based, quantitative potency assay that detects both transgenic expression and activity of an AAV8-hUGT1A1 vector, which is currently under clinical evaluation for the treatment of Crigler-Najjar syndrome. Potency of AAV8-hUGT1A1 was evaluated in vitro. After transduction of human hepatoma 7 (Huh7) cells, transgene-positive cells were quantified using flow cytometry and transgenic activity by a bilirubin conjugation assay. The in vitro potency of various AAV8-hUGT1A1 batches was compared with their potency in vivo. After AAV8-hUGT1A1 transduction, quantification of UGT1A1-expressing cells shows a linear dose-response relation (R² = 0.98) with adequate intra-assay and inter-day reproducibility (coefficient of variation [CV] = 11.0% and 22.6%, respectively). In accordance, bilirubin conjugation shows a linear dose-response relation (R² = 0.99) with adequate intra- and inter-day reproducibility in the low dose range (CV = 15.7% and 19.7%, respectively). Both in vitro potency assays reliably translate to in vivo efficacy of AAV8-hUGT1A1 vector lots. The described cell-based potency assay for AAV8-hUGT1A1 adequately determines transgenic UGT1A1 expression and activity, which is consistent with in vivo efficacy. This novel approach is suited for the determination of vector lot potency to support clinical-grade vector release.

• AAV8
• Crigler-Najjar syndrome
• UGT1A1 activity
• adeno-associated virus
• bilirubin
• gene therapy
• potency
• quantitative potency assay
• transduction efficiency

• Clinical burden
• Crigler-Najjar
• Mortality
• Phototherapy
• Transplantation

Adeno-associated virus (AAV) vector-mediated gene therapy is currently evaluated as a potential treatment for Crigler–Najjar syndrome (CN) (NCT03466463). Pre-existing immunity to AAV is known to hinder gene transfer efficacy, restricting enrollment of seropositive subjects in ongoing clinical trials. We assessed the prevalence of anti-AAV serotype 8 (AAV8) neutralizing antibodies (NAbs) in subjects affected by CN and investigated the impact of low NAb titers (<1:5) on liver gene transfer efficacy in an in vivo passive immunization model. A total of 49 subjects with a confirmed molecular diagnosis of CN were included in an international multicenter study (NCT02302690). Pre-existing NAbs against AAV8 were detected in 30.6% (15/49) of screened patients and, in the majority of positive cases, cross-reactivity to AAV2 and AAV5 was detected. To investigate the impact of low NAbs on AAV vector-mediated liver transduction efficiency, adult wild-type C57BL/6 mice were passively immunized with pooled human donor-derived immunoglobulins to achieve titers of up to 1:3.16. After immunization, animals were injected with different AAV8 vector preparations. Hepatic vector gene copy number was unaffected by low anti-AAV8 NAb titers when column-purified AAV vector batches containing both full and empty capsids were used. In summary, although pre-existing anti-AAV8 immunity can be found in about a third of subjects affected by CN, low anti-AAV8 NAb titers are less likely to affect liver transduction efficiency when using AAV vector preparations manufactured to contain both full and empty capsids. These findings have implications for the design of liver gene transfer clinical trials and for the definition of inclusion criteria related to seropositivity of potential participants.

• AAV gene therapy
• Crigler–Najjar syndrome
• UGT1A1
• anti-AAV neutralizing antibodies
• pre-existing immunity
• unconjugated hyperbilirubinemia

Various pathologic conditions result in jaundice, a yellowing of the skin due to a buildup of bilirubin. Patients with jaundice commonly report experiencing an intense non-histaminergic itch. Despite this association, the pruritogenic capacity of bilirubin itself has not been described, and no bilirubin receptor has been identified. Here, we demonstrate that pathophysiologic levels of bilirubin excite peripheral itch sensory neurons and elicit pruritus through MRGPRs, a family of G-protein coupled receptors expressed in primary sensory neurons. Bilirubin binds and activates two MRGPRs, mouse MRGPRA1 and human MRGPRX4. In two mouse models of pathologic hyperbilirubinemia, we show that genetic deletion of either Mrgpra1 or Blvra, the gene that encodes the bilirubin-producing enzyme biliverdin reductase, attenuates itch. Similarly, plasma isolated from hyperbilirubinemic patients evoked itch in wild-type animals but not Mrgpra1^-/- animals. Removing bilirubin decreased the pruritogenic capacity of patient plasma. Based on these data, targeting MRGPRs is a promising strategy for alleviating jaundice-associated itch.

Jaundice causes the skin to yellow as a result of a build-up of a pigment called bilirubin. Normally, bilirubin is made in the liver and removed from the body in digestive fluid called bile, but people with liver or gallbladder problems may end up with too much bilirubin that accumulates in their blood and skin. One side effect of jaundice is intense and uncontrollable itching. Researchers are not sure what causes this itching, and there are few treatments that help to relieve it.

At the molecular level, itching sensations occur when compounds bind to particular receptors on the surface of nerve cells. One family of receptors that can trigger itch is called the Mas-related G-protein Coupled Receptor (MRGPR). Could one of these receptors trigger jaundice-related itching?

Now, Meixiong, Vasavda et al. show that bilirubin binds to and activates MRGPRs to cause itch in mice. Whereas injecting bilirubin into normal mice causes them to scratch, mice that have been genetically engineered to lack MRGPRs do not itch when their own bilirubin levels rise, or when they are injected with bilirubin or with plasma from patients who experience jaundice-related itching. Furthermore, removing bilirubin from the plasma of patients before it was injected into normal mice reduced the amount of itching that the mice felt.

Overall, the results reported by Meixiong, Vasavda et al. suggest that drugs that prevent bilirubin from attaching to MRGPRs might help to alleviate jaundice-related itching. However, researchers must first verify that bilirubin interacts with MRGPRs in people to cause itch. If bilirubin causes itch in people like in mice, scientists could then evaluate existing drugs or make new ones to prevent bilirubin from attaching to the MRGPRs.

• Mrgpr
• bilirubin
• cholestasis
• itch
• mouse
• neuroscience
• sensory neuron

• AAV vector
• Crigler-Najjar syndrome
• UGT1A1
• liver gene transfer
• long-term safety

Crigler‐Najjar syndrome type 1 (CN1) is an autosomal recessive disease caused by a marked decrease in uridine‐diphosphate‐glucuronosyltransferase (UGT1A1) enzyme activity. Delivery of hUGT1A1‐modRNA (a modified messenger RNA encoding for UGT1A1) as a lipid nanoparticle is anticipated to restore hepatic expression of UGT1A1, allowing normal glucuronidation and clearance of bilirubin in patients. To support translation from preclinical to clinical studies, and first‐in‐human studies, a quantitative systems pharmacology (QSP) model was developed. The QSP model was calibrated to plasma and liver mRNA, and total serum bilirubin in Gunn rats, an animal model of CN1. This QSP model adequately captured the observed plasma and liver biomarker behavior across a range of doses and dose regimens in Gunn rats. First‐in‐human dose projections made using the translated model indicated that 0.5 mg/kg Q4W dose should provide a clinically meaningful and sustained reduction of >5 mg/dL in total bilirubin levels.

• Gilbert’s syndrome
• Isolated indirect hyperbilirubinemia
• Phenobarbitone

We aimed to identify potent biliverdin reductase (BVRA) inhibitors as a novel concept for the treatment of severe unconjugated hyperbilirubinemia. 1280 FDA-approved compounds were screened in vitro for their ability to inhibit human and rat BVRA activity and 26 compounds were identified as BVRA inhibitors. Montelukast and Disulfiram were selected as potentially clinically applicable drugs and tested to reduce serum unconjugated bilirubin (UCB) levels in the Ugt1a1-deficient rat, a model for chronic unconjugated hyperbilirubinemia. Oral administration of Disulfiram was toxic in the Ugt1a1-deficient rat (weight loss, transaminase elevation). Oral Montelukast administration led to low serum concentrations and did not alter serum UCB levels. Intraperitoneal injections of Montelukast resulted in concentrations up to 110 μmol/L in serum and 400 μmol/L in the liver. Still, serum UCB levels remained unaltered. This first study on biliverdin reductase inhibition as a novel concept for treatment of unconjugated hyperbilirubinemia identified putative in vitro BVRA inhibitors. Montelukast, the clinically most suitable inhibitor, did not result in reduction of serum UCB in the Ugt1a1-deficient rat. The proposed treatment strategy will not result in amelioration of severe unconjugated hyperbilirubinemia in humans without the identification or development of more potent BVRA inhibitors.

Crigler-Najjar syndrome is a severe metabolic disease of the liver due to a reduced activity of the UDP Glucuronosyltransferase 1A1 (UGT1A1) enzyme. In an effort to translate to the clinic an adeno-associated virus vector mediated liver gene transfer approach to treat Crigler-Najjar syndrome, we developed and optimized a vector expressing the UGT1A1 transgene. For this purpose, we designed and tested in vitro and in vivo multiple codon-optimized UGT1A1 transgene cDNAs. We also optimized noncoding sequences in the transgene expression cassette. Our results indicate that transgene codon-optimization is a strategy that can improve efficacy of gene transfer but needs to be carefully tested in vitro and in vivo. Additionally, while inclusion of introns can enhance gene expression, optimization of these introns, and in particular removal of cryptic ATGs and splice sites, is an important maneuver to enhance safety and efficacy of gene transfer. Finally, using a translationally optimized adeno-associated virus vector expressing the UGT1A1 transgene, we demonstrated rescue of the phenotype of Crigler-Najjar syndrome in two animal models of the disease, Gunn rats and Ugt1a1^-/- mice. We also showed long-term (>1 year) correction of the disease in Gunn rats. These results support further translation of the approach to humans.

Crigler-Najjar syndrome (CN) is a very rare genetic disorder characterized by an inability to conjugate bilirubin. Contrary to CN type I, patients with CN II exhibit residual capacity to conjugate bilirubin and may present a normal life expectancy.

We report an unusual late diagnosis of CN type II in an 80-year-old female admitted with severe acute cholangitis. While the patient present typical clinical and radiologic signs of bile duct obstruction and cholangitis, her blood analysis showed severe unconjugated hyperbilirubinemia. Endoscopic retrograde cholangiopancreatography confirmed the diagnosis and allowed therapeutic intervention. The anatomopathologic examination of her gallbladder following cholecystectomy showed signs of chronic cholecystitis.

The risk of gallstone disease may be increased in patients with CN syndrome. While unusual, we alert to this curious and potential life-threatening presentation.

• Acute cholangitis
• Choledocolitiasis
• Crigler Najjar
• Hyperbilirubinemia
• Uridine diphosphate-glucuronosyl-transferase deficiency

X-linked ichthyosis (XLI) is an X-linked recessive skin disorder generally restricted to males, which arises from mutations in the steroid sulfatase (STS) gene located on Xp22.3. Crigler-Najjar syndrome (CN-I) is a rare autosomal recessive disease caused by the homozygous or compound heterozygous mutations in the UPD-glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) gene on chromosome 2q37. A male patient was referred to the Department of Medical Genetics with of severe icterus and ichthyosis. The patient and his family members underwent genetic tests related to XLI and CN-I. Quantitative polymerase chain reaction on genomic DNA was performed to determine the gene copy number, while single nucleotide polymorphism array analysis was conducted to identify deletion mutations. Family pedigree analysis showed that the patient and his two cousins were all affected by ichthyosis, which was in accordance with the inheritance pattern of an X-linked recessive disease. In addition, the patient's serum bilirubin concentration (>340 mmol/l) was markedly greater than the normal level. The patient presented with kernicterus and phenobarbital treatment was ineffective. The clinical diagnosis of XLI was confirmed molecularly by laboratory evidence of a maternal 1.61 M deletion (including the STS gene) on ChrXp22.31. Coincidentally, the male patient was also confirmed to carry a rare maternal inherited microdeletion (374 Kb) comprising the entire UGT1A1 gene combined with a paternal UGT1A1 mutation (c.1253delT), a causative event of CN-I. To the best of our knowledge, this study reported for the first time the comorbidity of XLI and CN-I in a male patient. The results suggested that co-occurrence of these two recessive diseases in a patient may be incidental.

AIM: To test the ability of adult-derived human liver stem/progenitor cells (ADHLSC) from large scale cultures to conjugate bilirubin in vitro and in bilirubin conjugation deficient rat.

METHODS: ADHLSC from large scale cultures were tested for their phenotype and for their capacity to conjugate bilirubin in vitro after hepatogenic differentiation. In vivo, Gunn rats [uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1) deficient animal] were injected with ADHLSC and cryopreserved hepatocytes (positive control). Two, 4, 13 and 27 wk post-transplantation, transplanted Gunn rat bilirubin serum levels were determined by high performance liquid chromatography. Human transplanted cell engraftment was assessed 27 wk post-transplantation using immunohistochemistry and RTqPCR.

RESULTS: Large scale culture conditions do not modify ADHLSC phenotype, ADHLSC were able to specifically conjugate bilirubin. ADHLSC were intraportally injected into Gunn rats and blood UCB was measured at different times post-transplantation, infused-Gunn rats exhibited a metabolic effect 3 mo post-transplantation and maintained over a 6 mo period. ADHLSC engraftment into Gunn rat’s liver was demonstrated by RTqPCR and immunohistochemistry against albumin and UGT1A1.

CONCLUSION: ADHLSC from large scale cultures are efficient in conjugating bilirubin in vitro and in restoring a deficient metabolic function (reducing bilirubin level) in hyperbilirubinemic rats.

• Liver stem/progenitor cells
• Gunn rat
• Hepatocyte
• in vitro and in situ differentiation
• Uridine diphosphate-glucuronosyltransferase 1A1

• Animals
• Bilirubin/blood
• Biomarkers/blood
• Cell Differentiation
• Cells, Cultured
• Child
• Disease Models, Animal
• Down-Regulation
• Glucuronosyltransferase/deficiency
• Glucuronosyltransferase/genetics
• Glucuronosyltransferase/metabolism
• Hepatocytes/enzymology
• Hepatocytes/metabolism
• Hepatocytes/transplantation
• Humans
• Hypotension, Orthostatic/blood
• Hypotension, Orthostatic/diagnosis
• Hypotension, Orthostatic/enzymology
• Hypotension, Orthostatic/genetics
• Hypotension, Orthostatic/surgery
• Male
• Phenotype
• Rats, Gunn
• Serum Albumin/metabolism
• Stem Cell Transplantation
• Stem Cells/enzymology
• Stem Cells/metabolism
• Time Factors

• Bile Secretion
• Crigler–Najjar Syndrome
• Glucuronosyl Transferase
• Hepatic Storage Disease
• Kernicterus

• Genetic diseases
• Inborn
• Liver transplantation
• Metabolic diseases
• Rare diseases
• Surgical gene therapy

• Acidosis/surgery
• Humans
• Liver/enzymology
• Liver Diseases/surgery
• Liver Transplantation
• Methylmalonyl-CoA Decarboxylase/metabolism
• Propionic Acidemia/surgery
• Quality of Life
• Risk Assessment