Insulin-like growth factor binding protein 5 enhances survival of LX2 human hepatic stellate cells

ABSTRACT: BACKGROUND: Expression of insulin-like growth factor binding protein 5 (IGFBP5) is strongly induced upon activation of hepatic stellate cells and their transdifferentiation into myofibroblasts in vitro. This was confirmed in vivo in an animal model of liver fibrosis. Since IGFBP5 has been shown to promote fibrosis in other tissues, the aim of this study was to investigate its role in the progression of liver fibrosis. METHODS: The effect of IGFBP5 was studied in LX2 cells, a model for partially activated hepatic stellate cells, and in human primary liver myofibroblasts. IGFBP5 signalling was modulated by the addition of recombinant protein, by lentiviral overexpression, and by siRNA mediated silencing. Furthermore, the addition of IGF1 and silencing of the IGF1R was used to investigate the role of the IGF-axis in IGFBP5 mediated effects. RESULTS: IGFBP5 enhanced the survival of LX2 cells and myofibroblasts via a >50% suppression of apoptosis. This effect of IGFBP5 was not modulated by the addition of IGF1, nor by silencing of the IGF1R. Additionally, IGFBP5 was able to enhance the expression of established pro-fibrotic markers, such as collagen Ialpha1, TIMP1 and MMP1. CONCLUSION: IGFBP5 enhances the survival of (partially) activated hepatic stellate cells and myofibroblasts by lowering apoptosis via an IGF1-independent mechanism, and enhances the expression of profibrotic genes. Its lowered expression may, therefore, reduce the progression of liver fibrosi

Towards Liver-Directed Gene Therapy for Crigler-Najjar Syndrome

Crigler-Najjar (CN) syndrome is a recessive inherited disorder caused by deficiency of uridine diphosphoglucuronosyl 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 diseas

Progress and challenges in gene therapy for Crigler-Najjar syndrome

Introduction: Adeno-associated viral (AAV) vector-mediated, liver-directed gene therapy has shown to be feasible and effective to improve coagulopathy in patients with hemophilia B. Inherited severe unconjugated hyperbilirubinemia, known as Crigler-Najjar syndrome (CN), is a suitable disease model for AAV-mediated gene therapy, enabling application of this approach in the near future.Areas covered: This review provides an overview of gene therapy strategies for CN, focusing on AAV vector-mediated gene transfer. Despite good progress, major challenges regarding pre-existing humoral immunity against the vector and the inability to re-administer the viral vector have yet to be overcome. Here we discuss possible solutions for these challenges and cover other emerging therapeutic strategies for liver-directed gene therapy.Expert opinion: Promising results from pre-clinical studies warrant the translation of AAV vector-mediated gene therapy for the treatment of CN toward clinical application. Steps have been taken toward a phase I/II trial, while a screening protocol will reveal the prevalence of pre-existing humoral immunity in the CN patient population. Other emerging gene therapeutic strategies hold great promise, such as targeted genomic integration that may result in a more robust expression of the transgene or ex vivo gene integrating therapy followed by transplantation of the corrected cells. Both approaches still require extensive research before clinical application can be considered. Our focus for the near future rests upon the readily available AAV vector-mediated, liver-directed gene therapy platform for the treatment of Crigler-Najjar syndrom

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. A lack of effective treatment makes a liver transplantation necessary in most patients. In view of long-term adverse effects, for instance due to life-long immune suppression needed to prevent organ rejection, gene therapy could be a preferable approach, as supported by proof of concept in animal models for PFIC3. This review discusses the feasibility of gene therapy as an alternative for liver transplantation for all forms of PFIC based on their pathological mechanism. Conclusion: Using presently available gene therapy vectors, major hurdles need to be overcome to make gene therapy for all types of PFIC a reality

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 ad-dition, ABCB11 function is affected in 3 other types of PFIC. A lack of effective treatment makes a liver transplantation necessary in most patients. In view of long-term adverse effects, for instance due to life-long immune suppression needed to prevent organ rejec-tion, gene therapy could be a preferable approach, as supported by proof of concept in animal models for PFIC3. This review discusses the feasibility of gene therapy as an alter-native for liver transplantation for all forms of PFIC based on their pathological mecha-nism. Conclusion: Using presently available gene therapy vectors, major hurdles need to be overcome to make gene therapy for all types of PFIC a reality

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. A 3 nucleotide insertion in the HNF-1α binding site in the proximal promoter previously reported in a Crigler-Najjar patient on one allele and a novel two nucleotide deletion in exon 1, resulting in a frameshift and a premature stop codon. Conclusion: In newly diagnosed CNs patients with unconjugated bilirubin levels consistent with CNs type II but that are unresponsive to phenobarbital treatment, disruption of the HNF-1α binding site in the proximal promoter should be considered as a probable cause. Upon confirming a mutation in the HNF-1α site, phenobarbital treatment should be stopped or at least be reconsidered because of its sedative effects and its teratogenic properties

Mycophenolate Mofetil Impairs Transduction of Single-Stranded Adeno-Associated Viral Vectors

Adeno-associated virus (AAV) liver-directed gene therapy seems a feasible treatment for Crigler-Najjar syndrome type I, an inherited liver disorder characterized by severe unconjugated hyperbilirubinemia. Transient immunosuppression coupled with vector administration seems needed to overcome host immune responses that prevent long-term expression in patients. The immunosuppressive mycophenolate mofetil (MMF), which inhibits de novo synthesis of purines, is a promising candidate. To investigate the potential use of MMF in patients with Crigler-Najjar syndrome, we studied its effect on single-stranded AAV (ssAAV)-mediated correction of hyperbilirubinemia in the relevant preclinical model, the Gunn rat. Although MMF was well tolerated and effective it also impaired the efficacy of ssAAV. Subsequent in vitro studies showed that this effect is not specific for UGT1A deficiency. In fact, clinical relevant concentrations of mycophenolic acid (MPA), the active compound of MMF, also impair the transduction of HEK-293T cells by ssAAV. Because this effect was reversed by guanosine addition, it seems that intracellular levels of this nucleotide become limited, suggesting that MPA impairs second-strand DNA synthesis. This is corroborated by observations that MPA did not impair transduction of 293T cells by a self-complementary AAV (scAAV) vector and that MMF did not reduce the scAAV efficacy in the Gunn rat. In conclusion, MMF impairs ssAAV-mediated liver-directed gene therapy, which is relevant for the use of this immunosuppressive agent with single-stranded vectors. Furthermore, because this effect is due to impaired second-strand synthesis, the use of MMF with scAAV seems warrante

Crigler-Najjar syndrome in The Netherlands: Identification of four novel UGT1A1 alleles, 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. Hum Mutat 30:1-8, 2009. (c) 2009 Wiley-Liss, In