Inborn Errors Of Liver Metabolism Research Articles

Biomarkers for liver disease in urea cycle disorders

BackgroundUrea cycle disorders (UCDs) are among the most common inborn errors of liver metabolism. As therapies for hyperammonemia associated with urea cycle dysfunction have improved, chronic complications, such as liver disease, have become increasingly apparent in individuals with UCDs. Liver disease in UCDs may be associated with hepatic inflammation, hepatic fibrosis, portal hypertension, liver cancer and even liver failure. However, except for monitoring serum aminotransferases, there are no clear guidelines for screening and/or monitoring individuals with UCDs for liver disease. Thus, we systematically evaluated the potential utility of several non-invasive biomarkers for liver fibrosis in UCDs. MethodsWe evaluated grey-scale ultrasonography, liver stiffness obtained from shear wave elastography (SWE), and various serum biomarkers for hepatic fibrosis and necroinflammation, in a cohort of 28 children and adults with various UCDs. ResultsOverall, we demonstrate a high burden of liver disease in our participants with 46% of participants having abnormal grey-scale ultrasound pattern of the liver parenchyma, and 52% of individuals having increased liver stiffness. The analysis of serum biomarkers revealed that 32% of participants had elevated FibroTest™ score, a marker for hepatic fibrosis, and 25% of participants had increased ActiTest™ score, a marker for necroinflammation. Interestingly, liver stiffness did not correlate with ultrasound appearance or FibroTest™. ConclusionOverall, our results demonstrate the high overall burden of liver disease in UCDs and highlights the need for further studies exploring new tools for identifying and monitoring individuals with UCDs who are at risk for this complication. Trial registrationThis study has been registered in ClinicalTrials.gov (NCT03721367).

Challenges of experimental gene therapy for urea cycle disorders

Urea cycle disorders (UCDs) are inborn errors of liver metabolism that often result in hyperammonemia and failure of arginine synthesis due to the deficiencies of one of the six enzymes or two mitochondrial transporters involved. Despite current treatment options, including dietary therapy, stimulation of alternative routes of nitrogen disposal and cell therapies (hepatocyte and orthotopic liver transplantation), significant morbidity and mortality still remain. Gene therapy has emerged as an attractive alternative strategy for providing a definitive cure for patients with metabolic diseases. Successful phenotype correction in pre- clinical animal models is encouraging and research effort is increasingly being focused on translation from the laboratory bench to proof-of-concept human therapy. Gene therapy for UCDs must target the periportal hepatocytes, as these are the only liver cells that express all six enzymes required for full ammonia detoxification. This review explores current efforts to develop and translate liver-directed gene therapy approaches to UCDs caused by each of the defects of the six enzymes. The prominent issues that will need to be addressed and overcome for the future development of clinical trials, including alternatives to mouse mutants and to vectors that are not necessarily conditioned to rodents are also discussed.

Concise Review: Updated Advances and Current Challenges in Cell Therapy for Inborn Liver Metabolic Defects.

: The development of liver cell transplantation (LCT), considered a major biotechnological breakthrough, was intended to provide more accessible treatments for liver disease patients. By preserving the native recipient liver and decreasing hospitalization time, this innovative approach has progressively gained interest among clinicians. LCT initially targets inborn errors of liver metabolism, enabling the compensation of deficient metabolic functions for up to 18 months post-transplantation, supporting its use at least as a bridge to transplantation. The rigorous clinical development and widespread use of LCT depends strongly on controlled and consistent clinical trial data, which may help improve several critical factors, including the standardization of raw biological material and immunosuppression regimens. Substantial effort has also been made in defining and optimizing the most efficient cell population to be transplanted in the liver setting. Although isolated hepatocytes remain the best cell type, showing positive clinical results, their widespread use is hampered by their poor resistance to both cryopreservation and in vitro culture, as well as ever-more-significant donor shortages. Hence, there is considerable interest in developing more standardized and widely accessible cell medicinal products to improve engraftment permanency and post-cell transplantation metabolic effects. In this therapeutic approach to liver disease, new solutions are being designed and evaluated to bypass the documented limitations and move forward toward wide clinical use. Future developments also require a deep knowledge of regulatory framework to launch specific clinical trials that will allow clear assessment of cell therapy and help patients with significant unmet medical needs.

Treating inborn errors of liver metabolism with stem cells: current clinical development

Advanced therapies including stem cells are currently a major biotechnological development. Adult liver stem cells can differentiate into hepatocyte like cells and be infused in the recipient’s liver to bring a missing metabolic function. These cells can be produced in large quantities in vitro. Allogeneic stem cells are required to treat genetic diseases, and this approach allows to use one single source of tissue to treat different diseases and many recipients. Mesenchymal stem cells can in addition play an immunomodulatory and anti-inflammatory role and possibly prevent the accumulation of fibrous tissue in the liver. From a regulatory point of view, stem cells are considered as medicinal products, and must undergo a pharmaceutical development that goes beyond the research and proof-of-concept phases. Here, we review the track followed from the first hepatocyte transplantation in 2000 to the next generation product issued from stem cell technology, and the start of EMA approved clinical trials to evaluate the safety and potency of liver stem cells for the treatment of inborn errors of the liver metabolism.

A genome-scale modeling approach to study inborn errors of liver metabolism: toward an in silico patient.

Inborn errors of metabolism (IEM) are genetic diseases caused by mutations in enzymes or transporters affecting specific metabolic reactions that cause a block in the physiological metabolic fluxes. Therapeutic treatment can be achieved either by decreasing the metabolic flux upstream of the block or by increasing the flux downstream of the block. The identification of upstream and downstream fluxes however is not trivial, since metabolic reactions are intertwined in a complex network. To overcome this problem, we propose an innovative computational workflow to model the alteration of metabolism caused by IEM and predict the metabolites and reactions that are affected by the mutation. Our workflow exploits a recent genome-scale metabolic network model of hepatocyte metabolism to identify metabolites accumulating in hepatocytes due to single gene mutations in IEM via an innovative "differential flux analysis." We simulated 38 IEMs in the liver, and in about half of the cases, our workflow correctly identified the metabolites known to accumulate in the blood and urine of IEM patients.

In Vivo Selection of Transplanted Hepatocytes by Pharmacological Inhibition of Fumarylacetoacetate Hydrolase in Wild-type Mice

Genetic fumarylacetoacetate hydrolase (Fah) deficiency is unique in that healthy gene-corrected hepatocytes have a strong growth advantage and can repopulate the diseased liver. Unfortunately, similar positive selection of gene-corrected cells is absent in most inborn errors of liver metabolism and it is difficult to reach the cell replacement index required for therapeutic benefit. Therefore, methods to transiently create a growth advantage for genetically modified hepatocytes in any genetic background would be advantageous. To mimic the selective pressure of Fah deficiency in normal animals, an efficient in vivo small molecule inhibitor of FAH, 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate (CEHPOBA) was developed. Microarray analysis demonstrated that pharmacological inhibition of FAH produced highly similar gene expression changes to genetic deficiency. As proof of principle, hepatocytes lacking homogentisic acid dioxygenase (Hgd) and hence resistant to FAH inhibition were transplanted into sex-mismatched wild-type recipients. Time course analyses of 4-6 weeks of CEHPOBA administration after transplantation showed a linear relationship between treatment length and replacement index. Compared to controls, recipients treated with the FAH-inhibitor had 20-100-fold increases in liver repopulation. We conclude that pharmacological inhibition of FAH is a promising approach to in vivo selection of hepatocytes.

End‐stage Liver Disease and Liver Transplant: Current Situation and Key Issues

The aim of this article is to identify the current challenges in pediatric OLT. It is directed to all physicians who care for pediatric patients from the early prospect of liver transplant to posttransplant life. Besides assessing current information on transplant results, the coauthors of this article have identified a number of issues that they consider as the most important that need to be addressed in 2008. Some are only an update of old questions, and others are newly identified problems. The recommendations from the group are summarized at the end of each specific issue and in the consensus guidelines.

Gene therapy for inborn errors of liver metabolism: progress towards clinical applications

The treatment for inborn errors of liver metabolism is based on dietary, drug, and cell therapies (orthotopic liver transplantation). However, significant morbidity and mortality still remain, and alternative strategies are needed. Gene replacement therapy has the potential of providing a definitive cure for patients with these diseases. Significant progress has been made in the pre-clinical arena and achievement of efficacy in different animal models has been reported using multiple gene transfer technologies. This article summarizes the gene transfer strategies being investigated, the pre-clinical data, and the available early clinical results for inborn errors of liver metabolism.

Sustained Engraftment and Tissue Enzyme Activity After Liver Cell Transplantation for Argininosuccinate Lyase Deficiency

Donor cell engraftment with expression of enzyme activity is the goal of liver cell transplantation for inborn errors of liver metabolism with a view to achieving sustained metabolic control. Sequential hepatic cell transplantations using male and female cells were performed in a 3.5-year-old girl with argininosuccinate lyase deficiency over a period of 5 months. Beside clinical, psychomotor, and metabolic follow-up, engraftment was analyzed in repeated liver biopsies (2.5, 5, 8, and 12 months after first infusion) by fluorescence in situ hybridization for the Y-chromosome and by measurement of tissue enzyme activity. Metabolic control was achieved together with psychomotor catch-up, changing the clinical phenotype from a severe neonatal one to a moderate late-onset type. The child was no longer hospitalized and was able to attend normal school. Sustained engraftment of male donor liver cells was shown in repeated biopsies, reaching 19% at 8 months and 12.5% at the 12-month follow-up. XXYY tetraploid donor cells were mainly detected during the infusion period (2.5- and 5-month biopsies), whereas in the follow-up 8-month and 1-year biopsies, diploid donor cell subpopulations had become dominant. Moreover, argininosuccinate lyase activity, originally absent, became measurable in 2 different biopsy samples at 8 months, reaching 3% of control activity, indicating in situ metabolic effect and supporting the clinical evolution to a moderate form of the disease. Liver cell transplantation can achieve donor cell engraftment in humans in a significant proportion, leading to sustained metabolic and clinical control with psychomotor catch-up.

Hepatic proliferation in Gunn rats transplanted with hepatocytes: effect of retrorsine and tri‐iodothyronine

Hepatocyte transplantation would offer an attractive alternative to liver transplantation in the treatment of inborn errors of liver metabolism. However, a major problem in most transplantation studies to date has been the limited growth of transplanted cells in the recipient organ. We performed a strategy for selective proliferation of transplanted cells by interfering with the proliferative capacity of resident hepatocytes, using the pyrrolizidine alkaloid retrorsine and then transplanting liver cells in conjunction with repeated administration of triiodothyronine, an inducer of hepatocyte proliferation in rats. In the present study, foetal and adult syngeneic hepatocyte transplantation into spleen was performed in retrorsine-treated hyperbilirubinemic Gunn rats. In parallel, repeated injections of triiodothyronine were given to recipients. Rats were sacrificed at 1, 7, 30 and 90 days after transplantation and blood and bile samples were taken to assess the functionality of transplanted cells. The proliferative activity of transplanted hepatocytes was evaluated using proliferating cell nuclear antigen labelling index. In summary, both adult and foetal hepatocyte transplantation were effective in correcting a metabolic abnormality in Gunn rats for as long as 3 months. The RS/T3 model, as a measure to increase graft function, could represent an important advance to future clinical application of hepatocyte transplantation.

P0227 REPEATED LIVER CELL TRANSPLANTATION VIA PORTH A CATH SYSTEM IN A CHILD

Introduction: Liver cell transplantation is a novel experimental approach for the treatment of inborn errors of liver metabolism. We report a repeated cell infusion approach via a Porth A Cath system for the treatment of an eight year’s old girl with Crigler Najjar Syndrome. Methods: Hepatocytes were isolated from the left liver segment of one female and two male identical donors using the classical two step collagenase perfusion method. Cell recovery accounted for 3.8, 2.1, and 2.15 billions respectively, and viability ranged between 81 & 87 % (Trypan Blue exclusion method). A Porth A Cath system was inserted into a jejunal vein, and the access chamber placed subcutaneously in the left hypochondrion. 1.9 billion fresh isolated hepatocytes were first infused over two hours. The child received then five separate infusions for a total of 4.18 billion hepatocytes. The fourth first infusions used fresh liver cells, whereas cryopreserved cells were infused for the subsequent 5 infusions. The procedure was well tolerated, without any change in portal blood flow and portal pressure following the infusions. She received triple immunosuppression including anti IL2r antibodies (Simulect, Novartis, Bâle, Sw), tacrolimus (Fujisawa, Berlin, G) and steroids. Results: Transplantation efficiency was evaluated by measuring total Bilirubin levels, under identical phototherapy program. Mean bilirubin level during the five years before transplantation were 17.5±0.49 mg/dL. The level dropped to 16.16±0.31 mg/dL after the first infusions * p<0.05 and to 15.08±0.27 mg/dL after the second infusions. Six months after the first infusion, and the total of nine infusions, bilirubin levels had decreased progressively (13.6±0.42 mg/dL *** p<0.001) to reach the lowest value of 11.4 mg/dL. The effect of LCT was however not persistent, and bilirubin returned thereafter to pre- transplant values from month 6 post-LCT. The reason for the loss of metabolic effect could be rejection or apoptosis of transplanted cells. Conclusion: We conclude that liver cell transplantation can be done safely and repeatedly via a permanent portal access system, using a Porth A Cath. Metabolic effect in this case was temporary, which means that repopulation did not occur despite original efficient engraftment.

Recombinant human hepatocyte growth factor facilitates biliary transport after hepatocyte transplantation in Eisai hyperbilirubinemic rats.

Hepatocyte transplantation may offer an attractive treatment for inborn errors of liver metabolism. However, factor(s) are required as stimuli to induce proliferation of the limited number of hepatocytes transplanted. The Eisai hyperbilirubinemic rat (EHBR) is a Sprague-Dawley (SD) mutant rat with conjugated hyperbilirubinemia. EHBRs have impaired canalicular excretory transport of organic anions, bile acid glucuronide, and sulfate. Recombinant human hepatocyte growth factor (rhHGF) (100 microg/kg) was injected intravenously at 2-hr intervals for 10 hr, immediately and 35 days following the intraportal injection of 1 x 10(7) wild-type SD rat hepatocytes. Serum bilirubin concentrations decreased significantly within 35 days and were maintained at significantly reduced levels for 120 days following transplantation. Biliary excretion was demonstrated by the biliary transport of indocyanine green and sulfobromophthalein sodium into the bile. These results indicate that hepatic transport of bile acid conjugates in EHBRs can be restored by hepatocyte transplantation combined with repeated administration of exogenous rhHGF, in conjunction with functioning of the recipient's excretory biliary system.

42 COMPLEMENTATION OF THE METABOLIC DEFECT IN GUNN RAT HEPATOCYTES AFTER TRANSFECTION.

Most of the congenital errors of liver metabolism affect one single pathway and spare the general function and structure of the liver. Liver transplantation is a possible treatment, but the number of donor organs is limited and this therapy has a considerable rate of mortality. Gene therapy might be an alternative for the treatment of many inborn errors of liver metabolism. We study gene therapy for Crigler-Najjar disease type I. This disease is due to mutations in the UDP dependent glucuronyl transferase (B-UDPGT) in the liver. Plasmid based vectors were made and tested using a rat model (Gunn-rat) of the disease. For the gene transfer into primary rat hepatocytes the cationic liposome DOTAP™ (Boehringer Mannheim) and the synthetic peptide P2lip/HA (M Wilke et al submitted) were tested in transient transfection experiments. Efficient transfection of primary Gunn rat hepatocytes (20-30% LacZ +, > 106 U/mg luciferase) could be achieved under optimal conditions. The transfection efficiencies depended on the presence of mitogens in the culture medium with both transfection systems. This shows that synthetic peptides can be used for the development of gene transfer protocols into hepatocytes. For the complementation experiments in the Gunn-rat model we cloned three different expression vectors. We tested constructs with and without intron sequences as well as a liver-specific vector. After the transfection of Gunn rat hepatocytes complementation of the bilirubin conjugation defect could be shown by HPLC analysis. In these experiments the vector containing the intron showed the highest level of expression. The success of the in vitro experiments enables us to investigate the possibilities of in vivo gene transfer. In vivo gene transfer to the liver with plasmid vector depends on an efficient route of delivery, the targeting of the DNA/transfection complexes to parenchymal cells. Pilot experiments with fluorescent microspheres in the rat showed, that both retrograde perfusion of the bile duct and isolated perfusion through the portal vein are suitable routes of delivery. The advantage of peptide derived transfection compounds is, that they can be modified, e.g. by adding receptor ligands such as galactose. This ensures liver-specific gene expression in in vivo experiments.