Crigler-Najjar syndrome: looking to the future does not make us forget the present.

Recently, the safety and efficacy of gene therapy were evaluated in patients with Crigler-Najjar syndrome (CNS). Although it is a promising curative option for CNS, many doubts still persist about its long-term efficacy and safety. Furthermore, there is a risk of overlooking several unresolved problems still present in current clinical practice. This letter is a call for action on crucial open issues that remain nowadays an unmet need in the management of CNS patients.

Synthetic augmentation of bilirubin metabolism in human pluripotent stem cell-derived liver organoids.

UGT1A1 (UDP glucuronosyltransferase family 1 member A1) is the primary enzyme required for bilirubin conjugation, which is essential for preventing hyperbilirubinemia. Animal models lack key human organic anion transporting polypeptides with distinct epigenetic control over bilirubin metabolism, necessitating a human model to interrogate the regulatory mechanism behind UGT1A1 function. Here, we use induced pluripotent stem cells to develop human liver organoids that can emulate conjugation failure phenotype. Bilirubin conjugation assays, chromatin immunoprecipitation, and transcriptome analysis elucidated the role of glucocorticoid antagonism in UGT1A1 activation. This antagonism prevents the binding of transcriptional repressor MECP2 at the expense of NRF2 with associated off-target effects. Therefore, we introduced functional GULO (L-gulonolactone oxidase) in human organoids to augment intracellular ascorbate for NRF2 reactivation. This engineered organoid conjugated more bilirubin and protected against hyperbilirubinemia when transplanted in immunosuppressed Crigler-Najjar syndrome rat model. Collectively, we demonstrate that our organoid system serves as a manipulatable model for interrogating hyperbilirubinemia and potential therapeutic development.

Gene Therapy in Patients with the Crigler-Najjar Syndrome.

BACKGROUND: Patients with the Crigler-Najjar syndrome lack the enzyme uridine diphosphoglucuronate glucuronosyltransferase 1A1 (UGT1A1), the absence of which leads to severe unconjugated hyperbilirubinemia that can cause irreversible neurologic injury and death. Prolonged, daily phototherapy partially controls the jaundice, but the only definitive cure is liver transplantation. METHODS: We report the results of the dose-escalation portion of a phase 1-2 study evaluating the safety and efficacy of a single intravenous infusion of an adeno-associated virus serotype 8 vector encoding UGT1A1 in patients with the Crigler-Najjar syndrome that was being treated with phototherapy. Five patients received a single infusion of the gene construct (GNT0003): two received 2×1012 vector genomes (vg) per kilogram of body weight, and three received 5×1012 vg per kilogram. The primary end points were measures of safety and efficacy; efficacy was defined as a serum bilirubin level of 300 µmol per liter or lower measured at 17 weeks, 1 week after discontinuation of phototherapy. RESULTS: No serious adverse events were reported. The most common adverse events were headache and alterations in liver-enzyme levels. Alanine aminotransferase increased to levels above the upper limit of the normal range in four patients, a finding potentially related to an immune response against the infused vector; these patients were treated with a course of glucocorticoids. By week 16, serum bilirubin levels in patients who received the lower dose of GNT0003 exceeded 300 µmol per liter. The patients who received the higher dose had bilirubin levels below 300 µmol per liter in the absence of phototherapy at the end of follow-up (mean [±SD] baseline bilirubin level, 351±56 µmol per liter; mean level at the final follow-up visit [week 78 in two patients and week 80 in the other], 149±33 µmol per liter). CONCLUSIONS: No serious adverse events were reported in patients treated with the gene-therapy vector GNT0003 in this small study. Patients who received the higher dose had a decrease in bilirubin levels and were not receiving phototherapy at least 78 weeks after vector administration. (Funded by Genethon and others; ClinicalTrials.gov number, NCT03466463.).

Gilbert or Crigler-Najjar syndrome? Neonatal severe unconjugated hyperbilirubinemia with P364L UGT1A1 homozygosity.

BACKGROUND: Several mutations of bilirubin uridine diphosphate-glucuronosyltransferase gene (UGT1A1) have been reported in patients with unconjugated hyperbilirubinemia. Few reports are available about the p.Pro364Leu mutation (P364L, c.1091C > T) in homozygous newborns. We describe the clinical, laboratory and therapeutic approach in two Chinese neonates with severe jaundice, homozygous for the P364L mutation. CASE PRESENTATION: Two Chinese breastfed female infants presented prolonged unconjugated hyperbilirubinemia at the age of 1 month. Total bilirubin was higher than 15 mg/dl (D < 1). An exhaustive etiological work-up to detect possible causes of hyperbilirubinemia (notably hemolytic ones) was negative. The promoter and coding regions of UGT1A1 were amplified by polymerase chain reaction (PCR) from genomic DNA isolated from leukocytes. Both patients resulted homozygous for a variant site within the coding region of the gene in the 4 exon, c.1091C > T, p.Pro364Leu. In front of the persistently high level of unconjugated bilirubin, phototherapy was performed without persistent results. A treatment with phenobarbital was then begun and bilirubin level progressively decreased, with a complete and persistent normalization. The therapy was stopped. CONCLUSION: UGT1A1 enzyme activity associated with the P364L mutation has been described as 35.6% of the wild-type enzyme activity. Photo-therapy and phenobarbital can be useful in front of persistently high level of unconjugated bilirubin. Our cases presented high bilirubin values, overlapping between Gilbert syndrome (GS) and Crigler-Najjar syndrome type II (CNS), but the complete normalization of bilirubin makes GS more likely. Homozygous P364L variant can be associated with severe neonatal unconjugated hyperbilirubinemia in Chinese infants, but jaundice can completely resolve in a few months, contrary to what happens in Crigler-Najjar syndrome type II.

Induction of fecal cholesterol excretion is not effective for the treatment of hyperbilirubinemia in Gunn rats.

BACKGROUND: Unconjugated hyperbilirubinemia, a feature of neonatal jaundice or Crigler-Najjar syndrome, can lead to neurotoxicity and even death. We previously demonstrated that unconjugated bilirubin (UCB) can be eliminated via transintestinal excretion in Gunn rats, a model of unconjugated hyperbilirubinemia, and that this is stimulated by enhancing fecal fatty acid excretion. Since transintestinal excretion also occurs for cholesterol (TICE), we hypothesized that increasing fecal cholesterol excretion and/or TICE could also enhance fecal UCB disposal and subsequently lower plasma UCB concentrations. METHODS: To determine whether increasing fecal cholesterol excretion could ameliorate unconjugated hyperbilirubinemia, we treated hyperbilirubinemic Gunn rats with ezetimibe (EZE), an intestinal cholesterol absorption inhibitor, and/or a liver X receptor (LXR) and farnesoid X receptor (FXR) agonist (T0901317 (T09) and obeticholic acid (OCA), respectively), known to stimulate TICE. RESULTS: We found that EZE treatment alone or in combination with T09 or OCA increased fecal cholesterol disposal but did not lower plasma UCB levels. CONCLUSIONS: These findings do not support a link between the regulation of transintestinal excretion of cholesterol and bilirubin. Furthermore, induction of fecal cholesterol excretion is not a potential therapy for unconjugated hyperbilirubinemia. IMPACT: Increasing fecal cholesterol excretion is not effective to treat unconjugated hyperbilirubinemia. This is the first time a potential relation between transintestinal excretion of cholesterol and unconjugated bilirubin is investigated. Transintestinal excretion of cholesterol and unconjugated bilirubin do not seem to be quantitatively linked. Unlike intestinal fatty acids, cholesterol cannot "capture" unconjugated bilirubin to increase its excretion. These results add to our understanding of ways to improve and factors regulating unconjugated bilirubin disposal in hyperbilirubinemic conditions.

Human liver stem cells express UGT1A1 and improve phenotype of immunocompromised Crigler Najjar syndrome type I mice.

Crigler Najjar Syndrome type I (CNSI) is a rare recessive disorder caused by mutations in the Ugt1a1 gene. There is no permanent cure except for liver transplantation, and current therapies present several shortcomings. Since stem cell-based therapy offers a promising alternative for the treatment of this disorder, we evaluated the therapeutic potential of human liver stem cells (HLSC) in immune-compromised NOD SCID Gamma (NSG)/Ugt1-/- mice, which closely mimic the pathological manifestations in CNSI patients. To assess whether HLSC expressed UGT1A1, decellularised mouse liver scaffolds were repopulated with these cells. After 15 days' culture ex vivo, HLSC differentiated into hepatocyte-like cells showing UGT1A1 expression and activity. For the in vivo human cell engraftment and recovery experiments, DiI-labelled HLSC were injected into the liver of 5 days old NSG/Ugt1-/- pups which were analysed at postnatal Day 21. HLSC expressed UGT1A1 in vivo, induced a strong decrease in serum unconjugated bilirubin, thus significantly improving phenotype and survival compared to untreated controls. A striking recovery from brain damage was also observed in HLSC-injected mutant mice versus controls. Our proof-of-concept study shows that HLSC express UGT1A1 in vivo and improve the phenotype and survival of NSG/Ugt1-/- mice, and show promises for the treatment of CNSI.

Crigler-Najjar Syndrome Type 1: Pathophysiology, Natural History, and Therapeutic Frontier.

BACKGROUND AND AIMS: We describe the pathophysiology, treatment, and outcome of Crigler-Najjar type 1 syndrome (CN1) in 28 UGT1A1 c.222C>A homozygotes followed for 520 aggregate patient-years. APPROACH AND RESULTS: Unbound ("free") bilirubin (Bf ) was measured in patient sera to characterize the binding of unconjugated bilirubin (BT ) to albumin (A) and validate their molar concentration ratio (BT /A) as an index of neurological risk. Two custom phototherapy systems were constructed from affordable materials to provide high irradiance in the outpatient setting; light dose was titrated to keep BT /A at least 30% below intravascular BT binding capacity (i.e., BT /A = 1.0). Categorical clinical outcomes were ascertained by chart review, and a measure (Lf ) was used to quantify liver fibrosis. Unbound bilirubin had a nonlinear relationship to BT (R2  = 0.71) and BT /A (R2  = 0.76), and Bf as a percentage of BT correlated inversely to the bilirubin-albumin equilibrium association binding constant (R2  = 0.69), which varied 10-fold among individuals. In newborns with CN1, unconjugated bilirubin increased 4.3 ± 1.1 mg/dL per day. Four (14%) neonates developed kernicterus between days 14 and 45 postnatal days of life; peak BT  ≥ 30 mg/dL and BT /A ≥ 1.0 mol:mol were equally predictive of perinatal brain injury (sensitivity 100%, specificity 93.3%, positive predictive value 88.0%), and starting phototherapy after age 13 days increased this risk 3.5-fold. Consistent phototherapy with 33-103 µW/cm2 •nm for 9.2 ± 1.1 hours/day kept BT and BT /A within safe limits throughout childhood, but BT increased 0.46 mg/dL per year to reach dangerous concentrations by 18 years of age. Liver transplantation (n = 17) normalized BT and eliminated phototherapy dependence. Liver explants showed fibrosis ranging from mild to severe. CONCLUSION: Seven decades after its discovery, CN1 remains a morbid and potentially fatal disorder.

Disease burden of Crigler-Najjar syndrome: Systematic review and future perspectives.

BACKGROUND AND AIM: Crigler-Najjar syndrome (CNS) results from biallelic mutations of UGT1A1 causing partial or total loss of uridine 5'-diphosphate glucuronyltransferase activity leading to unconjugated hyperbilirubinemia and its attendant risk for irreversible neurological injury (kernicterus). CNS is exceedingly rare and has been only partially characterized through relatively small studies, each comprising between two and 57 patients. METHODS: A systematic literature review was conducted to consolidate data on the patient, caregiver, and societal burden of CNS. RESULTS: Twenty-eight articles on clinical aspects of CNS were identified, but no published data on its humanistic or economic burden were found. In patients with complete UGT1A1 deficiency (type 1 CNS [CNS-I]), unconjugated bilirubin levels increase 3-6 mg/dL/day during the newborn period and reach neurologically dangerous levels between 5 and 14 days of age. Phototherapy is the mainstay of treatment but poses significant challenges to patients and their families. Despite consistent phototherapy, patients with CNS-I have worsening hyperbilirubinemia with advancing age. Liver transplantation is the only definitive therapy for CNS-I and is increasingly associated with excellent long-term survival but also incurs high costs, medical and surgical morbidities, and risks of immunosuppression. CONCLUSIONS: Crigler-Najjar syndrome is associated with a substantial burden, even with existing standards of care. The development of novel disease-modifying therapies has the potential to reduce disease burden and improve the lives of CNS patients and their families.

Regenerative cell therapy for the treatment of hyperbilirubinemic Gunn rats with fresh and frozen human induced pluripotent stem cells-derived hepatic stem cells.

Pluripotent stem cells have been investigated as a renewable source of therapeutic hepatic cells, in order to overcome the lack of transplantable donor hepatocytes. Whereas different studies were able to correct hepatic defects in animal models, they focused on the most mature phenotype of hepatocyte-like cells (HLCs) derived from pluripotent stem cells and needed freshly prepared cells, which limits clinical applications of HLCs. Here, we report the production of hepatic stem cells (pHSCs) from human-induced pluripotent stem cells (hiPSCs) in xeno-free, feeder-free, and chemically defined conditions using as extracellular matrix a recombinant laminin instead of Matrigel, an undefined animal-derived matrix. Freshly prepared and frozen pHSCs were transplanted via splenic injection in Gunn rats, the animal model for Crigler-Najjar syndrome. Following cell transplantation and daily immunosuppression treatment, bilirubinemia was significantly decreased (around 30% decrease, P < .05) and remained stable throughout the 6-month study. The transplanted pHSCs underwent maturation in vivo to restore the deficient metabolic hepatic function (bilirubin glucuronidation by UGT1A1). In conclusion, we demonstrate for the first time the differentiation of hiPSCs into pHSCs that (a) are produced using a differentiation protocol compatible with Good Manufacturing Practices, (b) can be frozen, and (c) are sufficient to demonstrate in vivo therapeutic efficacy to significantly lower hyperbilirubinemia in a model of inherited liver disease, despite their immature phenotype. Thus, our approach provides major advances toward future clinical applications and would facilitate cell therapy manufacturing from human pluripotent stem cells.

Prevalence and Relevance of Pre-Existing Anti-Adeno-Associated Virus Immunity in the Context of Gene Therapy for Crigler-Najjar Syndrome.

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.

Quantitative Systems Pharmacology Model of hUGT1A1-modRNA Encoding for the UGT1A1 Enzyme to Treat Crigler-Najjar Syndrome Type 1.

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.

AAV8 Gene Therapy Rescues the Newborn Phenotype of a Mouse Model of Crigler-Najjar.

Adeno-associated viral (AAV) vectors can target the liver, making them an attractive platform for gene therapy approaches that require the correction of hepatocytes. Crigler-Najjar syndrome is an autosomal recessive disorder of bilirubin metabolism that occurs when the liver's uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) enzyme activity is partially or completely absent. This syndrome is characterized by elevated bilirubin levels in the blood. An AAV8 vector was developed expressing a codon-optimized human version of UGT1A1 from a liver-specific promoter. High doses of the vector rescued neonatal lethality in newborn UGT1 knockout (KO) mice, which serve as a model of Crigler-Najjar syndrome, and significantly increased survival from 5 to 270 days. Newborn UGT1 KO mice treated with AAV had serum total bilirubin levels that were 5.7 times higher than the levels seen in heterozygous and wild-type mice, likely due to dilution of vector genome copies (GC) in the liver resulting from a proliferation of hepatocytes during growth of the animal. The elevation in serum total bilirubin levels in adult UGT1 KO mice depended on the AAV8 vector dose. At doses <1011 GC/mouse, total bilirubin levels returned to those seen in phototherapy-rescued UGT1 KO mice. Mice injected with vector at 1011 or 3 × 1011 GC/mouse had sustained reduced total bilirubin levels throughout the duration of the study. When an AAV8 vector was re-administered in mice with elevated total bilirubin levels, serum total bilirubin levels decreased to wild-type levels (0.1-0.3 mg/dL) in mice that received a vector dose of 3 × 1012 GC/kg. Therefore, a low-level and likely transient decrease in serum total bilirubin during the first days of life is necessary for rescuing the lethal phenotype present in the neonatal UGT1 KO mouse. Furthermore, it was possible to ablate the elevated total bilirubin levels in adult mice by re-administering an AAV8 vector.

Crigler-Najjar Syndrome: Current Perspectives and the Application of Clinical Genetics.

BACKGROUND: Crigler-Najjar syndrome (CNS, OMIM: 218800) is the paradigm of an inborn error of metabolism and a rare genetic disease with an estimated incidence of 0.6-1.0 per million live births. Discrimination between CNS subtypes is usually done on the basis of the clinical criteria, such as response to phenobarbital treatment and other molecular and functional characteristics. METHODS: The identification of four novel pathogenic mutations and the analysis of residual activity of missense in UGT1A1 gene are useful for clinical diagnosis, and may reveal a new insight in enzyme activity, whereas the identification of pathogenic mutations will accelerate genetic counseling for newly identified CNS patients. RESULTS: Phototherapy, orthotropic liver transplantation, liver cell transplantation and gene therapy are treatment choices and candidates to fight back this syndrome. Due to the promising reports of gene therapy in small animal models, gene therapy approaches are expected to continue in preclinical research for developing safe and effective treatment of CNS. Gene transfer vectors using recombinant viruses, such as Adenovirus have been applied successfully in transferring UGT1A1 gene to the liver of Gunn rat model of CNS. CONCLUSION: In spite of remaining safety and efficiency issues, gene therapy promises to be a realistic treatment modality for CNS during the future decade.

Repeated AAV-mediated gene transfer by serotype switching enables long-lasting therapeutic levels of hUgt1a1 enzyme in a mouse model of Crigler-Najjar Syndrome Type I.

Adeno-associated virus (AAV) -mediated gene therapy is a promising strategy to treat liver-based monogenic diseases. However, two major obstacles limit its success: first, vector dilution in actively dividing cells, such as hepatocytes in neonates/children, due to the non-integrating nature of the vector; second, development of an immune response against the transgene and/or viral vector. Crigler-Najjar Syndrome Type I is a rare monogenic disease with neonatal onset, caused by mutations in the liver-specific UGT1 gene, with toxic accumulation of unconjugated bilirubin in plasma, tissues and brain. To establish an effective and long lasting cure, we applied AAV-mediated liver gene therapy to a relevant mouse model of the disease. Repeated gene transfer to adults by AAV-serotype switching, upon neonatal administration, resulted in lifelong correction of total bilirubin (TB) levels in both genders. In contrast, vector loss over time was observed after a single neonatal administration. Adult administration resulted in lifelong TB levels correction in male, but not female Ugt1-/- mice. Our findings demonstrate that neonatal AAV-mediated gene transfer to the liver supports a second transfer of the therapeutic vector, by preventing the induction of an immune response and supporting the possibility to improve AAV-therapeutic efficacy by repeated administration.

Promoterless gene targeting without nucleases rescues lethality of a Crigler-Najjar syndrome mouse model.

Crigler-Najjar syndrome type I (CNSI) is a rare monogenic disease characterized by severe neonatal unconjugated hyperbilirubinemia with a lifelong risk of neurological damage and death. Liver transplantation is the only curative option, which has several limitations and risks. We applied an in vivo gene targeting approach based on the insertion, without the use of nucleases, of a promoterless therapeutic cDNA into the albumin locus of a mouse model reproducing all major features of CNSI Neonatal transduction with the donor vector resulted in the complete rescue from neonatal lethality, with a therapeutic reduction in plasma bilirubin lasting for at least 12 months, the latest time point analyzed. Mutant mice, which expressed about 5-6% of WT Ugt1a1 levels, showed normal liver histology and motor-coordination abilities, suggesting no functional liver or brain abnormalities. These results proved that the promoterless gene therapy is applicable for CNSI, providing therapeutic levels of an intracellular ER membrane-bound enzyme responsible for a lethal liver metabolic disease.

[A 14-day-old boy with jaundice and apnoea]. / 14 dager gammel gutt med gulsott og apnéanfall.

We describe an infant who was readmitted from home at 14 days of age with jaundice and a history of apnoea and episodes of retrocollis/opisthotonos. He had been only mildly jaundiced on discharge from the maternity clinic at 2 days of age. The total serum bilirubin (TSB) on admission was 542 µmol/L, and the infant was treated intensively with triple phototherapy and exchange transfusion. In contrast to what is recommended in Norwegian national guidelines for management of neonatal jaundice, the parents had apparently neither received oral nor written information about jaundice and its follow-up at the time of discharge from maternity. They therefore contacted their child healthcare centre when they had questions about jaundice, though the national guidelines specifically state that follow-up for neonatal jaundice during the first 2 weeks of life is the responsibility of the birth hospital. Inappropriate advice resulted in delayed referral, and the child has been diagnosed with chronic kernicterus, probably the first such case in Norway since national guidelines were formalised in 2006. Genetic work-up disclosed compound heterozygosity for Crigler-Najjar syndrome type I, to the best of our knowledge the first instance of this disorder ever to have been diagnosed in Norway. The incidence of kernicterus is Norway is much lower than in other industrialised countries. This is most likely due to national guidelines for management of neonatal jaundice, which place the responsibility for management and follow-up of jaundice with the birth hospital during the crucial first 2 weeks of life. This case report reminds us that tragedies may occur when guidelines are disregarded.

Two Different UGT1A1 Mutations causing Crigler-Najjar Syndrome types I and II in an Iranian Family.

BACKGROUND: Crigler-Najjar syndrome type I (CN-1) and type II (CN-2) are rare hereditary unconjugated hyperbilirubinemia disorders. However, there have been no reports regarding the co-existence of CN-1 and CN-2 in one family. We experienced a case of an Iranian family that included members with either CN-1 or CN-2. Genetic analysis revealed a mutation in the bilirubin UDP-glucuronosyltransferase (UGT1A1) gene that resulted in residual enzymatic activity. CASE REPORT: The female proband developed severe hyperbilirubinemia [total serum bilirubin concentration (TB) = 34.8 mg/dL] with bilirubin encephalopathy (kernicterus) and died after liver transplantation. Her family history included a cousin with kernicterus (TB = 30.0 mg/dL) diagnosed as CN-1. Her great grandfather (TB unknown) and uncle (TB = 23.0 mg/dL) developed jaundice, but without any treatment, they remained healthy as CN-2. RESULTS: The affected cousin was homozygous for a novel frameshift mutation (c.381insGG, p.C127WfsX23). The affected uncle was compound heterozygous for p.C127WfsX23 and p.V225G linked with A(TA)7TAA. p.V225G-UGT1A1 reduced glucuronidation activity to 60% of wild-type. Thus, linkage of A(TA)7TAA and p.V225G might reduce UGT1A1 activity to 18%-36 % of the wild-type. CONCLUSION: Genetic and in vitro expression analyses are useful for accurate genetic counseling for a family with a history of both CN-1 and CN-2.

Amelioration of Hyperbilirubinemia in Gunn Rats after Transplantation of Human Induced Pluripotent Stem Cell-Derived Hepatocytes.

Hepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of hepatocyte-like cells (iHeps) differentiated from human induced pluripotent stem cells (iPSCs) could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1), where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%-7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%-60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases.