Repeated dosing of AAV-mediated liver gene therapy in juvenile rat and mouse models of Crigler-Najjar syndrome type I.

Crigler-Najjar syndrome is an ultra-rare monogenic recessive liver disease caused by gene mutations. Complete UGT1A1 deficiency results in severe unconjugated hyperbilirubinemia in newborns that, if not treated, may lead to brain damage and death. Treatment is based on intensive phototherapy, but its efficacy decreases with age, rendering liver transplantation the only curative option.

The Phospholipid Flippase ATP8B1 is Involved in the Pathogenesis of Ulcerative Colitis via Establishment of Intestinal Barrier Function.

Aims: Patients with mutations in ATP8B1 develop progressive familial intrahepatic cholestasis type 1 [PFIC1], a severe liver disease that requires life-saving liver transplantation. PFIC1 patients also present with gastrointestinal problems, including intestinal inflammation and diarrhoea, which are aggravated after liver transplantation. Here we investigate the intestinal function of ATP8B1 in relation to inflammatory bowel diseases.

Enhanced Visual Cortex Activation in People With Narcolepsy Type 1 During Active Sleep Resistance: An fMRI-EEG Study.

The brain activation patterns related to sleep resistance remain to be discovered in health and disease. The maintenance of wakefulness test (MWT) is an objective neuropsychological assessment often used to assess an individual's ability to resist sleep. It is frequently used in narcolepsy type 1, a disorder characterized by impaired sleep-wake control and the inability to resist daytime sleep.

Long-Term Effects of Biliverdin Reductase a Deficiency in Mice: Impact on Redox Status and Metabolism.

Accumulation of neurotoxic bilirubin due to a transient neonatal or persistent inherited deficiency of bilirubin glucuronidation activity can cause irreversible brain damage and death. Strategies to inhibit bilirubin production and prevent neurotoxicity in neonatal and adult settings seem promising. We evaluated the impact of deficiency in neonatal and aged mice, in a background of unconjugated hyperbilirubinemia, by abolishing bilirubin production.

Low efficacy of recombinant SV40 in Ugt1a1-/- mice with severe inherited hyperbilirubinemia.

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.

Reduced spontaneous itch in mouse models of cholestasis.

Pruritus is one of the most distressing symptoms in cholestatic patients. Plasma autotaxin (ATX) activity correlates with the severity of pruritus in cholestatic patients, but the pathophysiology is unclear. To study pruritus in mice, we measured scratch activity in cholestatic Atp8b1 mutant mice, a model for Progressive Familial Intrahepatic Cholestasis type 1, and wild type mice (WT) with alpha-naphthylisothiocyanate (ANIT)-induced cholestasis.

Efficacy of AAV8-h with Rapamycin in neonatal, suckling, and juvenile rats to model treatment in pediatric CNs patients.

A clinical trial using adeno-associated virus serotype 8 (AAV8)-human uridine diphosphate glucuronosyltransferase 1A1 (h) 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-h, and serum bilirubin levels and anti-AAV8 neutralizing antibodies (NAbs) in serum were monitored.

Gene Therapy for Progressive Familial Intrahepatic Cholestasis: Current Progress and Future Prospects.

Progressive Familial Intrahepatic Cholestasis (PFIC) are inherited severe liver disorders presenting early in life, with high serum bile salt and bilirubin levels. Six types have been reported, two of these are caused by deficiency of an ABC transporter; ABCB11 (bile salt export pump) in type 2; ABCB4 (phosphatidylcholine floppase) in type 3. In addition, ABCB11 function is affected in 3 other types of PFIC.

An Exon-Specific Small Nuclear U1 RNA (ExSpeU1) Improves Hepatic OTC Expression in a Splicing-Defective / Mouse Model of Ornithine Transcarbamylase Deficiency.

splicing mutations are generally associated with the severest and early disease onset of ornithine transcarbamylase deficiency (OTCD), the most common urea cycle disorder. Noticeably, splicing defects can be rescued by spliceosomal U1snRNA variants, which showed their efficacy in cellular and animal models. Here, we challenged an U1snRNA variant in the OTCD mouse model (/) carrying the mutation c.

A Quantitative Potency Assay for Adeno-Associated Virus Vectors Encoding for the Transgene.

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-h vector, which is currently under clinical evaluation for the treatment of Crigler-Najjar syndrome. Potency of AAV8-h was evaluated .

A Compensatory U1snRNA Partially Rescues FAH Splicing and Protein Expression in a Splicing-Defective Mouse Model of Tyrosinemia Type I.

The elucidation of aberrant splicing mechanisms, frequently associated with disease has led to the development of RNA therapeutics based on the U1snRNA, which is involved in 5' splice site (5'ss) recognition. Studies in cellular models have demonstrated that engineered U1snRNAs can rescue different splicing mutation types. However, the assessment of their correction potential in vivo is limited by the scarcity of animal models with the targetable splicing defects.

Steroidogenic control of liver metabolism through a nuclear receptor-network.

Objective: Coupling metabolic and reproductive pathways is essential for the survival of species. However, the functions of steroidogenic enzymes expressed in metabolic tissues are largely unknown.

Methods And Results: Here, we show that in the liver, the classical steroidogenic enzyme Cyp17a1 forms an essential nexus for glucose and ketone metabolism during feed-fast cycles.

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 passive immunization model.

A novel UGT1A1 gene mutation causing severe unconjugated hyperbilirubinemia: a case report.

Background: Crigler-Najjar syndrome (CNs) presents as unconjugated hyperbilirubinemia, as a result of UGT1A1 deficiency, and can be categorized in a severe (type I) and mild (type II) phenotype. CNs type II patients usually benefit from phenobarbital treatment that induces residual UGT1A1 activity.

Case Presentation: Here we present a CNs type II patient that is not responsive to phenobarbital treatment, which can be explained by two heterozygous mutations in the UGT1A1 gene.

The P4-ATPase ATP9A is a novel determinant of exosome release.

Extracellular vesicles (EVs) released by cells have a role in intercellular communication to regulate a wide range of biological processes. Two types of EVs can be recognized. Exosomes, which are released from multi-vesicular bodies upon fusion with the plasma membrane, and ectosomes, which directly bud from the plasma membrane.

Liver-directed gene therapy results in long-term correction of progressive familial intrahepatic cholestasis type 3 in mice.

Background & Aims: Progressive familial intrahepatic cholestasis type 3 (PFIC3), for which there are limited therapeutic options, often leads to end-stage liver disease before adulthood due to impaired ABCB4-dependent phospholipid transport to bile. Using adeno-associated virus serotype 8 (AAV8)-mediated gene therapy, we aimed to restore the phospholipid content in bile to levels that prevent liver damage, thereby enabling stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3.

Methods: Ten-week-old Abcb4 mice received a single dose of AAV8-hABCB4 (n = 10) or AAV8-GFP (n = 7) under control of a liver specific promoter via tail vein injection.

Cre Recombinase Mediates the Removal of Bacterial Backbone to Efficiently Generate rSV40.

Gene therapy has been shown to be a feasible approach to treat inherited disorders . Among the currently used viral vector systems, adeno-associated virus (AAV) vectors are the most advanced and have been applied in patients successfully. An important drawback of non-integrating AAV vectors is their loss of expression upon cell division, while repeating systemic administration lacks efficacy due to the induction of neutralizing antibodies.

Biliverdin Reductase inhibitors did not improve severe unconjugated hyperbilirubinemia in vivo.

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.