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Crigler-Najjar syndrome is an ultra-rare monogenic recessive liver disease caused by UGT1A1 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. Adeno-associated virus (AAV)-mediated gene therapy has shown long-term correction in adult patients, but loss of viral DNA and therapeutic efficacy are expected in younger patients associated with liver growth. Effective vector re-administration is hindered by anti-AAV neutralizing antibodies generated during the first administration. Here, we investigated AAV vector re-administration by modulating the immune response with rapamycin-loaded nanoparticles (ImmTOR) in Gunn rats ( Ugt1a -/- ) and Ugt1a -/- mice. We administered a liver-specific AAV8 vector expressing a codon-optimized h UGT1A 1 cDNA (1.0E11 vg/kg) in P25-P28 mutant animals and, upon loss of efficacy after 3 to 5 weeks, a higher second dose (1.0E12 or 5.0E12 vg/kg) was given. ImmTOR co-administration reduced anti-AAV neutralizing antibodies and immunoglobulin Gs generation in male animals of both models allowing effective re-dosing, underscored by a significant and long-term decrease in plasma bilirubin, although efficacy was affected by low-titer residual anti-AAV antibodies suggesting that re-administration in patients may require combination with other methods., Competing Interests: T.K.K. is an employee and shareholder of Selecta Bioscience; F.M., G.R., F.C., G.B., and A.F.M. are inventors in patents describing the AAV technology and gene therapy-based treatments for Crigler-Najjar syndrome., (© 2024 The Authors.)

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., Methods: ATP8B1 expression was investigated in intestinal samples of patients with Crohn's disease [CD] or ulcerative colitis [UC] as well as in murine models of intestinal inflammation. Colitis was induced in ATP8B1-deficient mice with dextran sodium sulphate [DSS] and intestinal permeability was investigated. Epithelial barrier function was assessed in ATP8B1 knockdown Caco2-BBE cells. Co-immunoprecipitation experiments were performed in Caco2-BBE cells overexpressing ATP8B1-eGFP. Expression and localization of ATP8B1 and tight junction proteins were investigated in cells and in biopsies of UC and PFIC1 patients., Results: ATP8B1 expression was decreased in UC and DSS-treated mice, and was associated with a decreased tight junctional pathway transcriptional programme. ATP8B1-deficient mice were extremely sensitive to DSS-induced colitis, as evidenced by increased intestinal barrier leakage. ATP8B1 knockdown cells showed delayed barrier establishment that affected Claudin-4 [CLDN4] levels and localization. CLDN4 immunohistochemistry showed a tight junctional staining in control tissue, whereas in UC and intestinal PFIC1 samples, CLDN4 was not properly localized., Conclusion: ATP8B1 is important in the establishment of the intestinal barrier. Downregulation of ATP8B1 levels in UC, and subsequent altered localization of tight junctional proteins, including CLDN4, might therefore be an important mechanism in UC pathophysiology., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

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., Competing Interests: The authors have declared that no competing interests exist.

Adamant progression of chronic cholangiopathies towards cirrhosis and limited therapeutic options leave a liver transplantation the only effective treatment. Insulin-like growth factor 1 (IGF1) effectively blocks fibrosis in acute models of liver damage in mice, and a phase I clinical trial suggested an improved liver function. IGF1 targets the biliary epithelium, but its potential benefit in chronic cholangiopathies has not been studied. To investigate the possible therapeutic effect of increased IGF1 expression, we crossed Abcb4(-/-) mice (a model for chronic cholangiopathy), with transgenic animals that overexpress IGF1. The effect on disease progression was studied in the resulting IGF1-overexpressing Abcb4(-/-) mice, and compared to that of Abcb4(-/-) littermates. The specificity of this effect was further studied in an acute model of fibrosis. The overexpression of IGF1 in transgenic Abcb4(-/-) mice resulted in stimulation of fibrogenic processes - as shown by increased expression of Tgfß, and collagens 1, 3 and 4, and confirmed by Sirius red staining and hydroxyproline measurements. Excessive extracellular matrix deposition was favored by raise in Timp1 and Timp2, while a reduction of tPA expression indicated lower tissue remodeling. These effects were accompanied by an increase in expression of inflammation markers like Tnfα, and higher presence of infiltrating macrophages. Finally, increased number of Ck19-expressing cells indicated proliferation of biliary epithelium. In contrast to liver fibrosis associated with hepatocellular damage, IGF1 overexpression does not inhibit liver fibrogenesis in chronic cholangiopathy., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A comprehensive understanding of the mechanisms that underlie hepatic differentiation inside a bioartificial liver (BAL) device is obtained when functional, histological, and gene expression analyses can be combined. We therefore developed a novel cell-sampling technique that enabled us to analyze adherent hepatocytes inside a BAL device during a 5-day culture period, without the necessity of terminating the culture. Biochemical data showed that hepatocyte-specific functions were relatively stable, despite an increase in glycolytic activity. Quantitative reverse transcriptase polymerase chain reaction analysis of hepatic genes cytochrome p450 3A29, albumin, glutamine synthetase, alpha-1 antitrypsin, and carbamoyl-phosphate synthetase, but also de-differentiation marker pi-class glutathione S transferase showed stable messenger ribonucleic acid (mRNA) levels from day 1 to 5. In contrast, mRNA levels of alpha-fetoprotein, pro- and anti-apoptotic genes Bax-alpha and Bcl-X(L), metabolic genes lactate dehydrogenase and uncoupling protein 2, and cytoskeleton genes alpha- and beta-tubulin and beta-actin increased in 5 days. Histological analysis revealed viable tissue-like structures with adaptation to the in vitro environment. We conclude that hepatocytes show a tendency for de-differentiation shortly after seeding but thereafter remain acceptably differentiated during 5 days of culture. Furthermore, partly impaired mitochondrial function is suggestive for local hypoxic regions and may trigger the observed metabolic changes. Anti-apoptotic activity seems to balance pro-apoptotic activity. This new cell-sampling technique facilitates the analysis of dynamic processes of hepatocyte culture inside a BAL.