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"The role of Glutamine synthetase in Liver Failure: Molecular, Functional and Therapeutic modulation"

Final Report Summary - HE&GS (The role of Glutamine synthetase in Liver Failure: Molecular, Functional and Therapeutic modulation)

Project Summary

Background and Aims
Hepatic encephalopathy (HE) is a complex disorder characterized by gradual impairment of the ability to perform mental tasks and to react to external stimuli, which can lead to coma and death. HE results from inefficient clearance by the liver of ammonia due to liver disease. As an example of the impact in health, cirrhosis is the major cause of chronic liver dysfunction and affects 1 million Europeans and 5.5million Americans.

An effective treatment for HE does not exist: ammonia is the central target of therapy but there are no interventions that have been shown to reduce ammonia predictably in liver failure. We suggest that greater understanding of the regulatory pathways involved in development of HE will lead to an effective therapy.
The project HE&GS provided an innovative and original approach to understand one of the key regulatory pathways of ammonia metabolism, the enzyme glutamine synthetase (GS). GS produces the amino acid glutamine removing ammonia from the circulation. We observed in a model of chronic liver failure that after the administration of OP (ornithine phenylacetate; a new molecule which decreases ammonia) GS increased in the muscle.
Question this project was designed to answer
• To investigate if GS is important in liver failure and
• If its enhancement (by re-insertion of the gene) in the muscle will have a positive impact in liver failure.
Question 1. To investigate if GS is important in liver failure
Studies in a muscle GS knock out animal
GS produces glutamine, which is an amino acid that is important in regulating ammonia metabolism, gut integrity, protein synthesis and immune function. Before the patients can be benefited by this treatment the first steps we needed were to check the lack of muscle GS in liver failure models. These experiments will find if there is enough data to support this treatment and build a construct containing the GS gene. We wanted a model in which GS in the muscle is missing, so, for the development of HE&GS we started an international collaboration with Professors Lamers and Harvort in the University of Amsterdam which provided us with a knock-out mouse with the selective deficiency of GS in the muscle.
Role of muscle GS in acute liver failure: In this model we were able to identify that in liver failure induced by paracetamol (commonest cause of acute liver failure), the severity of the illness was much higher. The animals lacking muscle GS had significantly greater brain swelling and liver injury that was associated with more severe hepatic inflammation. The mechanism of this was found to be due to the greater migration of bacteria from the gut into the blood stream.
These data suggested that replacement of muscle GS may be useful to reduce the severity of liver injury in this model.
Role of muscle GS in cachexia of cirrhosis: We observed that the knock-out animals had less muscle mass after receiving a special amino acid diet mimicking the composition of the blood (gastrointestinal haemorrhage is another problem in the patients with liver failure). Using mass spectrometric techniques in another model of liver failure (bile duct ligation) we identified that the animals lacking muscle GS had more severe oxidative stress in the muscle proteins and energy storage deficiency, which could explain, why the lack of GS function in the muscle produced muscle loss. As muscle depletion is frequently encountered in patients with cirrhosis and together with malnutrition is an independent prognostic factor for survival in cirrhosis, reconstitution of GS in the muscle is likely to be an important therapy.

These data suggested that replacement of muscle GS may be useful to reduce the muscle cachexia in a model of cirrhosis.

Question 2. Developing a gene therapy approach to replace muscle GS
After several attempts at preparing the genetic construct, we have now been able to successfully develop a viral construct that can be used to increase the amount of GS in the muscle using a safe virus. We aimed in our initial proposal to perform experiments to replace muscle GS. With this construct, it is now possible to produce a virus that can be used.
Conclusions
The results of this project has allowed further clarification of the important role of muscle GS and the associated mechanisms of the reduction of GS function and its effects in liver failure. Further development of the novel gene therapy construct will allow the generation of a new therapeutic approach for patients with hyperammonemia, liver failure and hepatic encephalopathy.

IMPACT
HE&GS project brings the opportunity of supporting the important role of glutamine synthetase as a target in liver failure, hepatic encephalopathy and its complications such as depletion of muscle mass and impairment in the gut barrier.
Healthcare impact: The results of this study are likely to have widespread impact for potential treatment of patients with acute liver failure and cirrhosis. Strategies to enhance muscle GS are likely to reduce severity of liver failure in the acute setting and reduce muscle loss in the chronic setting.
Economic impact: One of the main reasons why patients with cirrhosis require prolonged hospital admission and develop recurrent infections is depletion of muscle mass. The demonstration that the reduced muscle GS accounts for this liver disease related cachexia, provides a novel solution to the problem.
Novel therapy and potential new intellectual property: Further development of the gene therapy approach to replacement of muscle GS may allow generation of new IP which will have enormous impact for further development of this new therapeutic approach.