Use of glutamine synthetase to treat hyperammonemia - Patent Application 20070233633

Abstract

To provide glutamine synthase fusion protein for treating hyperammonemia, a pharmaceutical composition containing the fusion protein, and use of the fusion protein.SOLUTION: The present invention relates to use of glutamine synthase as protein therapy for treating hyperammonemia (such as enzyme replacement protein therapy). Particularly, the present invention relates to systemic administration of glutamine synthase. The glutamine synthase can be provided in a conjugate type or a fusion type in order to increase its half-life period during circulation. A pharmaceutical composition containing the glutamine synthase is also provided. The present invention also relates to use, a method and a composition including a combination of glutamine synthase protein and ammonia lowering agent such as nitrogen scavenger.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the use of glutamine synthetase as a protein therapy (such as enzyme replacement protein therapy) for the treatment of hyperammonemia, and in particular to the systemic administration of glutamine synthetase. Glutamine synthetase is thus administered as a protein or polypeptide, with the aim of increasing the circulating level of glutamine synthetase. Glutamine synthetase can be provided in a conjugated or fused form to increase its half-life in circulation. Pharmaceutical compositions comprising glutamine synthetase are also provided. The present invention also relates to uses, methods and compositions comprising the combination of glutamine synthetase protein and an ammonia-lowering agent, such as a nitrogen scavenger. [Background technology]

[0002] Hyperammonemia is a metabolic condition characterized by increased or excess ammonia in the blood. It is a dangerous condition primarily because it can lead to increased migration of ammonia to the brain, which in turn causes potentially extremely severe neurological and neuropsychiatric disorders leading, among others, to brain damage, seizures, retardation, coma, and even death. In fact, encephalopathy is a common and dangerous complication of hyperammonemia. The exact mechanism of encephalopathy / brain damage is yet to be elucidated, but it is believed that osmotic stress of astrocytes due to increased ammonia plays a role, leading to cerebral edema and increased intracranial pressure. Hyperammonemia can be congenital or acquired, and primary or secondary.

[0003] Primary (congenital) hyperammonemia results from a variety of inborn errors of metabolism characterized by reduced activity of any of the enzymes or transporter proteins of the urea cycle. In fact, such inborn errors of metabolism form a group of diseases called urea cycle disorders (UCDs). Ammonium (NH), in its charged form, is released into the blood in response to pH. 4 + ) and ammonia (NH 3) is a product of the catabolism of proteins and other nitrogenous compounds. Ammonia is converted to the less toxic substance, urea, by enzymes of the urea cycle before excretion in urine by the kidneys. The urea cycle, which also serves as the sole source of production of certain amino acids (arginine, citrulline and ornithine) in the body, contains six enzymes [five catalytic enzymes, carbamoyl phosphate synthase I (CPS1), ornithine transcarbamylase (OTC), or argininosuccinate synthase (ASS1), argininosuccinate lyase (ASL) and arginase (ARG), as well as the co-factor producing enzyme N-acetylglutamate synthase (NAGS)], and two transporter proteins, ornithine translocase (ORNT1) and citrine. Deficiencies can occur in any one or more of these eight proteins. Severe deficiency or complete absence of activity of any of the first four enzymes or of NAGS results in accumulation of ammonia and other precursor metabolites during the first few days of life. Infants with severe UCD are normal at birth but rapidly develop cerebral edema and associated signs of lethargy, anorexia, hyperventilation or hypoventilation, hyperthermia, seizures, neurological posturing, and coma. The severity of UCD is influenced by the location of the defective enzyme in the pathway and the severity of the enzyme deficiency. Rapid identification and current treatment strategies have dramatically improved survival rates of newborns with hyperammonemia in the past few decades, but intellectual abilities are typically impaired. In milder or partial deficiencies of these enzymes, as well as ARG deficiency, ammonia accumulation can be triggered by illness or stress at almost any time during life. In these disorders, elevated plasma ammonia concentrations and symptoms are often more subtle than the neonatal presentation of UCD, and the first recognized clinical episode may not occur for months or decades.

[0004] Secondary hyperammonemia results from inborn errors of intermediary metabolism characterized by decreased activity of enzymes / proteins that are not part of the urea cycle (e.g., propionic acidemia, methylmalonic acidemia, galactosemia, fatty acid oxidation disorders and mitochondrial disorders) or by impaired function of cells that contribute significantly to ammonia and / or nitrogen metabolism more broadly (e.g., the liver).

[0005] Acquired hyperammonemia usually results from liver disease, including both acute and chronic liver failure, such as viral hepatitis or excessive alcohol intake. Liver dysfunction or hepatic vascular bypass resulting in decreased hemofiltration in the liver leads to hyperammonemia. Hepatic encephalopathy due to hyperammonemia is a common complication of liver disease.

[0006] Hyperammonemia can also occur for other reasons, including renal dysfunction, e.g., renal dysfunction and / or renal failure, drug toxicity (e.g., with valproic acid or cyclophosphamide), idiopathic hyperammonemic syndrome following immunosuppressive or cytotoxic therapy, urea breakdown in retained urine and urinary tract infections, or essential amino acid total parenteral nutrition.

[0007] Current treatments for hyperammonemia are designed to reduce ammonia levels in the blood and / or brain, for example, by hemodialysis (commonly used in neonates) or by administering compounds that increase the removal of nitrogenous waste products, such as non-absorbable disaccharides (e.g., lactulose) or antibiotics (e.g., rifaxamin) or compounds that convert nitrogen to products other than urea, which are then excreted (e.g., compounds such as sodium benzoate, arginine, carglumic acid, phenylacetate or, more recently, phenylbutyrate, or L-ornithine L-aspartate (LOLA) or L-ornithine phenylacetate (OP)).

[0008] Management of the condition may also include dietary control to restrict protein intake and ensure adequate nutritional intake, including management of protein and / or nitrogen intake and parenteral intake of calories. Summary of the Invention [Problem to be solved by the invention]

[0009] However, despite improvements in the treatment and management of the condition, current therapies are non-specific and do not always manage the condition well.In particular, for UCD, current therapies cannot prevent many ammonia-elevating events, and patients with severe forms of the disease are often evaluated for liver transplantation at around 5 years of age.Therefore, there is a continuing need for additional or improved therapies for hyperammonemia. [Means for solving the problem]

[0010] The present invention seeks to address this need and is based on the concept of using glutamine synthetase to detoxify ammonia by converting it into the non-toxic product glutamine. In particular, the present invention proposes the systemic administration of glutamine synthetase as a protein therapy to reduce the level of ammonia in the blood.

[0011] Glutamine synthetase (GS) catalyzes the reaction: Glutamate + ATP + NH 3 →Glutamine + ADP + phosphate.

[0012] Glutamine synthesis occurs in several organs of the body and may play a role in organ and whole-body nitrogen balance. Very recently, a gene therapy based on overexpression of GS in skeletal muscle was proposed for the treatment of acute hyperammonemia (Torres-Vega et al, Gene Therapy 2015, 22, 58-64). The rationale for this therapy is to replace or increase GS, which is commonly deficient in the muscles of patients with liver disease, and thus to increase the clearance of ammonia by this enzyme in the muscles. However, gene therapy has proven difficult to successfully implement in clinical practice, and not all patients are suitable for gene therapy (e.g., children, with the exception of stem cell-based gene therapy), or patients may be refractory to gene therapy (e.g., for immune reasons). Moreover, such therapy would have a mainly local effect on the muscle. Thus, there remains a need for more generally applicable therapies.

[0013] Systemic administration of GS as a protein may help achieve such a more general effect. The inventors have shown that GS, particularly human GS, can be successfully expressed and purified, and can retain or exert GS activity in both unmodified and modified forms bound to polymeric partners such as polyethylene glycol. Furthermore, animal studies have shown that high circulating levels of GS can be achieved by systemic administration, and that both modified and unmodified GS administered to animals retain activity in blood and other tissues (e.g., liver). Thus, therapeutic levels of GS can be achieved by systemic (e.g., parenteral) administration of GS protein.

[0014] Furthermore, the inventors have also surprisingly found that the combination of a GS protein and an ammonia-lowering agent (such as a nitrogen scavenger, e.g., a pharma- ceutically acceptable salt of phenylacetic acid, such as sodium phenylacetate) has a synergistic effect, further enhancing the ability of the GS protein to treat or prevent hyperammonemia. Thus, in one embodiment, the present invention provides a glutamine synthetase (GS) protein for use in treating or preventing hyperammonemia by systemic parenteral administration to a subject.

[0015] In suitable embodiments, the GS protein for use in the treatment or prevention of hyperammonemia may be for use in combination with an ammonia-lowering agent.

[0016] In another aspect, the present invention provides an ammonia-lowering agent for use in combination with a GS protein for use in the treatment or prevention of hyperammonemia.

[0017] A related aspect of the invention also provides the use of a GS protein for the manufacture of a composition (e.g., a pharmaceutical or nutritional composition, such as a medicine or supplement) for the treatment or prevention of hyperammonemia by systemic parenteral administration to a subject.

[0018] In suitable embodiments, the compositions may be for use in combination with an ammonia reducing agent.

[0019] A further aspect of the present invention also provides the use of an ammonia-lowering agent for the manufacture of a composition for use in combination with a GS protein for the treatment or prevention of hyperammonemia.

[0020] In a further aspect, the present invention provides the use of a GS protein and an ammonia-reducing agent for the manufacture of a composition for use in the treatment or prevention of hyperammonemia.

[0021] In a further aspect, the present invention provides a method of treating or preventing hyperammonemia in a subject, comprising systemically and parenterally administering to said subject (more particularly to a subject in need thereof) a GS protein. In suitable embodiments, the method further comprises administering an ammonia-lowering agent.

[0022] Also provided is a composition comprising a GS protein for use in the treatment or prevention of hyperammonemia by systemic parenteral administration to a subject.

[0023] Suitably, the composition comprising the GS protein may be a pharmaceutical or nutritional composition, such as a medicine or a supplement. Suitably, the composition may further comprise an ammonia lowering agent.

[0024] The present invention also relates to a composition comprising an ammonia-reducing agent for use in the treatment or prevention of hyperammonemia. Suitably, the composition comprising an ammonia-reducing agent may be a pharmaceutical or nutritional composition, such as a medicine or a supplement.

[0025] The present invention also relates to compositions comprising a GS protein and an ammonia-lowering agent.

[0026] Suitably the composition may be a pharmaceutical or nutritional composition. Suitably the composition may be for use in the treatment or prevention of hyperammonemia.

[0027] The term "GS protein" can alternatively be expressed as a "protein having glutamine synthetase (GS) activity." The term "protein" is used broadly herein to include any proteinaceous molecule, including peptides and polypeptides, as well as protein or polypeptide fragments. As described in more detail below, the GS protein need not be or correspond to the full-length GS enzyme as it occurs in nature (e.g., native or wild-type GS), but includes truncated or other mutants. Also included are conjugates or fusions of the GS protein with other molecules, also as described in more detail below.

[0028] The term "hyperammonemia" includes any condition in which ammonia in the blood (or as measured or determined in any blood-derived product or sample, e.g., plasma) is elevated compared to the level of ammonia in a subject without any condition, e.g., a healthy subject, or a subject without an underlying disease that leads to or causes hyperammonemia. In health, ammonia transport and metabolism are tightly controlled to maintain low plasma / blood concentrations (normal range 10-40 μmol / L). Thus, plasma (or blood) ammonia concentrations of >40, 60 or 70 or 80 μmol / L or more, e.g., 41, 42, 45, 50, 55, 60, 70 or 80 μmol / L or more, may be considered indicative of hyperammonemia. For example, plasma ammonia concentrations of >100 μmol / L, and particularly 150 μmol / L or more, associated with a normal anion gap and normal plasma glucose concentrations, may indicate the presence of hyperammonemia, or, more specifically, UCD.

[0029] Hyperammonemia may result from any of the causes or conditions discussed above, i.e. hyperammonemia may be congenital or acquired, primary or secondary, as discussed above.Thus, in one embodiment, hyperammonemia may result from (or be associated with) urea cycle disorder (UCD).As discussed above, UCD may result from any one or more defects in the proteins of the urea cycle, which may inactivate or reduce the activity of the protein.

[0030] In a further embodiment, hyperammonemia may result from an inborn error of metabolism affecting a protein (e.g. an enzyme) that is not part of the urea cycle but that affects nitrogen metabolism and / or balance in the body, leading to an increase in the amount of ammonia in the blood. Suitably, such an inborn error of metabolism may be glutamine synthetase deficiency.

[0031] In further embodiments, hyperammonemia may be acquired and may result from disease or damage to an organ or tissue of the body involved in nitrogen metabolism and / or balance, e.g., catabolism and / or excretion of nitrogen-containing molecules or substances, e.g., the liver or kidney.

[0032] Thus, any type of liver injury or disease, including both chronic or acute liver failure, for example liver injury due to excessive alcohol consumption or due to drugs (whether recreational or pharmaceutical), cirrhosis from any cause, non-alcoholic fatty liver disease, liver infection or trauma to the liver can lead to hyperammonemia.

[0033] Similarly, any type of injury or disease of the kidney, as described above for the liver, can also lead to hyperammonemia. Thus, for example, any condition that affects multiple organs of the body (e.g., multiple organ failure), such as sepsis, organ damage from injury (whether from external injury, e.g., trauma, or internal injury, e.g., from an autoimmune disorder), or any systemic infection, can result in hyperammonemia.

[0034] Hyperammonemia can be detected or diagnosed based on clinical, biochemical and / or molecular genetic data, depending on the underlying cause. Thus, for example, hyperammonemia can be detected by evaluating or monitoring the blood level of ammonia (e.g., in plasma or serum or any blood-derived sample) according to techniques known and used in the art. Analysis of amino acids and / or their concentrations (e.g., arginine or citrulline) present in blood (such as plasma or serum), or other metabolites (e.g., urinary orotic acid) in blood or other body fluids or tissues may also help to identify that UCD is involved and / or determine its exact nature (i.e., the specific protein / enzyme deficiency involved). Such determination and analysis can be combined with clinical evaluation, such as neurological and neuropsychiatric evaluation, including both physical (e.g., MRI or other imaging) and / or behavioral / response tests, liver and / or kidney or other organ function tests, etc. If UCD is suspected, a family history investigation and / or molecular genetic tests and / or evaluation of the enzymatic activity of urea cycle enzymes may also be performed.

[0035] As used herein, reference to glutamine synthetase or GS protein for use according to the invention includes reference to all forms of enzymatically active GS, including human GS and GS from non-human animals (such as mouse, cow, rabbit, rat, monkey, chimpanzee, dog, etc.) or from other sources including, for example, fungi, plants or bacteria, as well as enzymatically active mutants. Representative GS proteins thus include those having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the GS polypeptide set forth in SEQ ID NO: 1 or 2 or 4 (human GS, precursor, mature N-terminally tagged form, respectively), or to the GS polypeptide set forth in SEQ ID NO: 6 [GS from Lactobacillus acididophilus strain 30SC] or SEQ ID NO: 7 [GS from maize, Zea Mays], or an enzymatically active fragment thereof. For example, reference to GS can also include N- and / or C-terminal truncated polypeptides or amino acid modified proteins (e.g., post-translational modifications such as adenylation, or other modifications such as amino acid polymorphisms that may affect the structure or activity of the protein). Reference to GS may also include multimers of the protein. The term "GS" therefore includes all naturally occurring GS enzymes or polypeptides, as well as enzymatically active fragments or variants thereof, including synthetically derived and modified polypeptides having one or more amino acid substitutions, additions (including insertions and extensions), or deletions that retain GS enzyme activity.

[0036] GS may therefore be or be derived from any enzyme within the scope of enzyme classification EC6.3.1.2. GS may be any polypeptide or peptide with GS activity. GS activity may be defined as the ability to convert glutamate and ammonia to glutamine, for example according to the reaction scheme shown above. GS activity may be assessed or determined using assays or tests (e.g. functional activity assays) known in the art and described in the literature. For example, GS enzyme activity assays are described in Listrom et al, Biochem. J. 1997, 328, 159-163. GS activity assays are also described in the following examples (see Examples 2 and 4).

[0037] The term also includes prodrugs of GS, which are forms that do not themselves exhibit GS activity, but which can be converted to active GS upon administration to a subject.

[0038] Thus, as used herein, "enzymatically active" with respect to a GS protein or polypeptide refers to a GS protein or polypeptide that is capable of catalyzing the conversion of glutamate and ammonia to glutamine. Typically, an enzymatically active GS protein or polypeptide exhibits at least or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the enzymatic activity of a GS polypeptide set forth in SEQ ID NO:1, 2, 4, 6 or 7.

[0039] The term "subject" as used herein includes any human or non-human animal, and particularly refers to a mammal, including, for example, humans, primates, livestock animals (e.g., sheep, pigs, cows, horses, donkeys), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs), companion animals (e.g., dogs, cats) and captive wild animals (e.g., foxes, kangaroos, deer).Preferably, the mammal is a human or a laboratory test animal.Even more preferably, the mammal is a human.

[0040] As used herein, the terms "treating", "treatment", "preventing" and "prevention" refer to any use of curing or ameliorating a condition or symptom, preventing the establishment of a condition or disease, or otherwise preventing, hindering, slowing, reducing or reversing in any way the progression of a condition or disease or other undesirable symptoms. Thus, the terms "treating" and "preventing" etc. should be considered in their broadest context. For example, treatment does not necessarily imply that the patient is treated until complete recovery, but includes any improvement or amelioration of the patient's or subject's condition, or symptoms of a disease or condition. Thus, for example, in the case of UCD, treatment according to the present invention does not of course treat the underlying genetic disorder, but rather treats the clinical condition resulting from hyperammonemia. In conditions exhibiting or characterized by multiple symptoms, treatment or prevention may not necessarily cure, ameliorate, prevent, hinder, delay, reduce or reverse all of said symptoms, but may cure, ameliorate, prevent, hinder, delay, reduce or reverse one or more of said symptoms. In suitable embodiments, "treatment" according to the invention may include reducing the level of ammonia in the blood, for example to a normal or healthy level, for example to the range of 10-40 μmol / L. Thus, treatment includes restoring normal or healthy ammonia levels. Similarly, prevention according to the invention may include maintaining plasma / blood ammonia levels in any normal or healthy range, as indicated above.

[0041] In suitable embodiments, "treatment" according to the present invention may include increasing glutamine synthetase levels and / or activity in a tissue of a subject, e.g., to normal or healthy levels. Such an increase may be in any suitable tissue, e.g., liver and / or muscle. An increase in glutamine synthetase levels and / or activity may be determined, for example, by measuring the glutamine level, glutamate level, and / or determining the ratio of glutamine level to glutamate level in a subject. It will be understood that normal or healthy glutamine and / or glutamate levels may vary depending on the sample in which they are measured. It will also be understood that normal or healthy glutamine and / or glutamate levels are subject-specific and may depend on factors such as the weight, diet, sex, and age of the subject. Normal or healthy glutamine and / or glutamate levels will be known to those skilled in the art.

[0042] In suitable embodiments, "treatment" according to the present invention may include reduction of edema. The term "edema" as used herein refers to abnormal accumulation of serous fluid in a subject. In suitable embodiments, edema may be cerebral edema, pulmonary edema, peripheral edema, and / or macular edema. Suitably, in the context of the present invention, "treatment" may refer to reduction of cerebral edema (e.g., of the prefrontal cortex). By way of example, edema can be assessed by CT scan, MRI, and / or x-ray. Other methods of assessing edema will be known to those skilled in the art.

[0043] In suitable embodiments, "treatment" according to the present invention may include the improvement of neuropsychological, neuropsychiatric and neurocognitive functions. The term "neuropsychological, neuropsychiatric and neurocognitive functions" refers to the brain functions that control, for example, memory, attention, cognition, psychomotor activity, coordination and mood. The uses, methods and compositions of the present invention, including the combination of GS protein and ammonia-lowering agents, may be particularly useful in such embodiments. Various methods for assessing neuronal function will be known to those skilled in the art. Neuronal functional status can be assessed using electroencephalography, computerized tests, paper-and-pencil tests, and evaluation by a neuropsychologist.

[0044] In a suitable embodiment, "treatment" according to the present invention can include sarcopenia and improvement of physical function. The term "sarcopenia" refers to the reduction of muscle mass. The term "physical function" refers to the strength of a subject, particularly muscle strength. The severity of sarcopenia can be determined by using clinical tools, nutritional tools, body composition measurements, or imaging. Physical function can be evaluated, for example, by oxygen delivery and consumption, exercise tests and grip strength tests, and / or improvement of fatigue level, and / or evaluation of immune system function.

[0045] As used herein, "amelioration" refers to a decrease in the severity of at least one indicator or symptom of a condition or disease. In certain embodiments, improvement includes a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of an indicator can be determined by subjective or objective measures known to those skilled in the art.

[0046] As used herein, the term "associated with" when used in the context of a disease or condition "associated with" elevated ammonia levels means that the disease or condition may result from, may lead to, may be characterized by, or may otherwise be associated with elevated ammonia levels. Thus, the association of a disease or condition with elevated ammonia levels may be direct or indirect, and may be separated in time.

[0047] Suitable samples for determining the level of ammonia include any suitable or desired sample in which ammonia may be generated.Similarly, suitable samples for determining glutamine synthetase level and / or glutamine synthetase activity include any suitable or desired sample in which glutamine synthetase, glutamine and / or glutamic acid may be present.They may be any suitable or desired tissue or body fluid sample.Examples of suitable tissues are liver and / or muscle tissue.Conveniently, the sample may be any body fluid sample, typically blood or any blood-derived sample, such as plasma or serum, but may also be any other body fluid, such as urine, cerebrospinal fluid, or feces or tissue sample, such as a biopsy sample or a lavage or washing fluid sample.This may of course depend on the exact nature of the condition to be treated, etc.

[0048] As used herein, the term "effective amount" includes within its meaning a non-toxic but sufficient amount or dose of GS protein and / or ammonia-reducing agent, depending on the context, to provide the desired effect. It will be understood that the effective amount of the protein and / or ammonia-reducing agent may vary. Exemplary therapeutically effective amounts are set forth elsewhere herein.

[0049] The exact amount or dose required will vary from subject to subject, depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular FMO3 being administered, and the mode of administration, etc. Thus, it is not feasible to specify an exact "effective amount." However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0050] Human GS is expressed as a 373 amino acid polypeptide (set forth in SEQ ID NO:1). This corresponds to the complete "precursor" protein when expressed, which is then further processed to a mature form having amino acids 2-373 (with only the N-terminal methionine removed to yield a 372 amino acid mature protein in vivo, set forth in SEQ ID NO:2). An exemplary polynucleotide set forth in SEQ ID NO:3 corresponds to a cDNA encoding the polypeptide of SEQ ID NO:1. SEQ ID NO:4 corresponds to a modified human GS protein comprising the GS polypeptide of SEQ ID NO:1 with an N-terminal His tag and linker sequence, as prepared and used in the examples below. SEQ ID NO:5 is a cDNA sequence encoding the polypeptide of SEQ ID NO:4 that has been codon-optimized for expression in bacteria, as used in the examples below.

[0051] Human GS has been well characterized (e.g., Listrom et al., 1997, see above). GS enzymes from other organisms, including plants and bacteria, have also been identified, and the nucleic acid and amino acid sequences of such other GS enzymes are well known in the art and are provided in freely available databases, such as the National Center for Biotechnology Information (NCBI) nucleotide (ncbi.nlm.nih.gov / nuccore) and protein (ncbi.nlm.nih.gov / protein) databases. Although the sequence identity between plant or bacterial GS enzymes and human GS may be low, the structural and functional similarity is high. Thus, plant or bacterial GS, or indeed GS from other organisms, or its amino acid sequence variants may be used. As representative examples, SEQ ID NO:6 sets forth the amino acid sequence of GS from Lactobacillus acidophilus strain 30SC, which has 23.8% sequence identity with human GS and 61.9% sequence identity with GS of Lactobacillus casei, and SEQ ID NO:7 sets forth the amino acid sequence of GS from corn (maize, Zea mays), which has 55.7% sequence identity with human GS.

[0052] GS generally exists as a multimer comprising multiple (i.e., 2 or more) monomeric subunits. For example, the GS amino acid sequence provided above corresponds to such a monomeric subunit. Human GS is most frequently reported as a dodecamer (12 subunits). As used herein, GS may be provided as a monomer and / or multimer. A multimer may comprise two or more monomeric subunits, for example, 2-20, 2-16, 2-15, 2-14, or 2-12 subunits.

[0053] Indeed, a surprising feature of the present invention is that, contrary to literature reports of 12 subunit multimers, human GS can be expressed and / or obtained as a mixture of multiple different types of multimers, including monomeric forms. The monomeric forms have been shown to be active. Thus, according to the present invention, GS can be used as a monomer and / or as a multimer, and the multimers can be provided as a single multimeric form or as a mixture of different multimeric forms that may or may not contain monomers. As reported in the examples below, 4 or more multimeric forms can be obtained, for example 4 to 10, for example 5 to 8. The size of the multimers can be 2 to 20 subunits.

[0054] The GS protein used in the methods provided herein can be obtained by any method known in the art, such as recombinant methods, protein isolation and purification methods, and chemical synthesis methods, provided that the GS obtained exhibits enzymatic activity. Thus, GS can be recombinant GS, natural GS isolated from tissue, or chemically synthesized GS.

[0055] It is well within the capabilities of one of ordinary skill in the art to modify a GS polypeptide, such as the polypeptide set forth in SEQ ID NO:1, to generate an enzymatically active GS variant for use in the methods provided herein. For example, one of ordinary skill in the art will understand that modifications at positions involved in substrate binding or the active site may be less tolerable than modifications at positions outside of these critical regions. Any GS polypeptide can be tested using methods well known in the art, such as those described in the Examples below, to assess the ability of the GS polypeptide to catalyze the conversion of glutamate and ammonia to glutamine.

[0056] In some examples, the GS used in accordance with the invention herein is a recombinant GS produced using prokaryotic or eukaryotic expression systems known in the art. Exemplary prokaryotic expression systems include, but are not limited to, the Escherichia coli expression system, and exemplary eukaryotic expression systems include, but are not limited to, yeast, insect cell, and mammalian cell expression systems.

[0057] Nucleic acid encoding GS can be obtained by any suitable method, including but not limited to RT-PCR of liver RNA and synthetic nucleotide synthesis. Primers for amplification can be designed based on known GS sequences, such as those described above. Nucleic acid and amino acid sequences of GS are well known in the art and are provided in freely available databases, such as the National Center for Biotechnology Information (NCBI) nucleotide (ncbi.nlm.nih.gov / nuccore) and protein (ncbi.nlm.nih.gov / protein) databases.

[0058] A nucleic acid encoding a GS polypeptide, such as a nucleic acid having a sequence set forth in SEQ ID NO:3, can be cloned into an expression vector suitable for an expression system of choice. In some instances, the nucleic acid is codon-optimized for expression in a particular system. For example, a nucleic acid encoding a GS polypeptide may be codon-optimized for expression in Escherichia coli. An exemplary codon-optimized nucleic acid encoding a GS polypeptide for expression in Escherichia coli is set forth in SEQ ID NO:5, which encodes a GS polypeptide including a His tag attached via a GGGGS linker (set forth in SEQ ID NO:4).

[0059] Typically, the nucleic acid encoding GS is cloned into an expression vector operably linked to a control sequence that facilitates the expression of a heterologous nucleic acid molecule. Many expression vectors suitable for the expression of GS are available and known to those skilled in the art. The selection of the expression vector is influenced by the selection of the host expression system. Such a selection is well within the level of skill of the skilled artisan. In general, the expression vector can include a transcription promoter and optionally an enhancer, a translation signal, and a transcription and translation termination signal. The expression vector used for stable transformation typically has a selectable marker that allows the selection and maintenance of transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector in the cell.

[0060] GS polypeptides can also be expressed as protein fusions. For example, fusions can be generated to add additional functionality to the polypeptide. Examples of fusion proteins include, but are not limited to, fusions containing GS and an affinity tag for purification (e.g., a His tag, such as his6, MYC, FLAG, HA or GST tag), a leader sequence (such as the pelB leader sequence), a sequence that directs protein secretion, or a protein that stabilizes and / or solubilizes GS (e.g., maltose binding protein (MBP)), or a protein that increases in vivo half-life (e.g., albumin or an Fc domain, or fragments thereof).

[0061] Prokaryotes, particularly E. coli, provide a system for producing large amounts of GS. Transformation of E. coli is a simple and rapid procedure well known to those skilled in the art. E. coli expression vectors can contain inducible promoters that are useful for inducing high levels of protein expression and for expressing proteins that exhibit some degree of toxicity to the host cell. Examples of inducible promoters include the lac promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters, and the temperature-regulated λPL promoter.

[0062] In other examples, eukaryotic expression systems such as baculovirus expression systems are used to generate GS. Typically, expression vectors use promoters such as the baculovirus polyhedrin promoter for high-level expression. Commonly used baculovirus systems include baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV), as well as insect cell lines such as Sf9 from Spodoptera frugiperda, Pseudaletia unipuncta (A7S) and Danaus plexippus (DpNl). For high-level expression, the GS nucleotide sequence is fused immediately downstream of the viral polyhedrin initiation codon.

[0063] Yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Kluyveromyces lactis, and Pichia pastoris can also be used as expression hosts for GS. Yeast can be transformed with episomal replicating vectors or by stable chromosomal integration by homologous recombination. Typically, inducible promoters such as GAL1, GAL7, and GAL5 are used to control gene expression. Yeast expression vectors often contain selectable markers such as LEU2, TRP1, HIS3, and URA3 for selection and maintenance of transformed DNA.

[0064] Mammalian expression systems can also be used to express GS. Expression constructs can be introduced into mammalian cells by viral infection, such as adenovirus, or by direct DNA transfer, such as liposomes, calcium phosphate, DEAE-dextran, and physical means, such as electroporation and microinjection. Expression vectors for mammalian cells typically contain an mRNA cap site, a TATA box, a translation initiation sequence (Kozak consensus sequence) and a polyadenylation element. Such vectors often contain transcriptional promoter-enhancers for high-level expression, such as the SV40 promoter-enhancer, the human cytomegalovirus (CMV) promoter, and the long terminal repeat of Rous sarcoma virus (RSV). Exemplary cell lines available for mammalian expression include, but are not limited to, mouse, rat, human, monkey, and chicken and hamster cells such as BHK, 293-F, CHO, Balb / 3T3, HeLa, MT2, mouse NSO (non-secreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2 / 0, COS, NIH3T3, HEK293, 293S, 293T, 2B8, and HKB cells.

[0065] After expression, GS can be purified using any method known to those skilled in the art, including but not limited to SDS-PAGE, size fractionation and size exclusion chromatography, ammonium sulfate precipitation, chelating chromatography, ion exchange chromatography and affinity chromatography. Affinity purification methods can be used to improve the efficiency and purity of the preparation. For example, antibodies and other molecules that bind GS can be used in affinity purification. As discussed above, the expression construct can be engineered to add affinity tags such as his, myc, FLAG or HA tags or GST moieties to GS, and then affinity purified using Ni resin, myc antibody, HA antibody, FLAG antibody or glutathione resin, respectively. Purity can be assessed by any method known in the art, including gel electrophoresis and staining and spectrophotometry, such as SDS-PAGE and size exclusion chromatography (SEC).

[0066] For use according to the invention, the affinity tag (such as a his tag) may be removed, although this is not necessary and the GS polypeptide may be used with the tag attached.

[0067] The tag or other fusion partner may be attached to the GS via a linker, which may be any suitable linker, according to principles well known in the art. Such linkers may typically and advantageously be short (e.g. 2-10, 2-8 or 2-6 mer) peptides. An example may be the linker GGSG, but may be composed of any suitable amino acids. Amino acid linkers allow the preparation of fusion proteins by recombinant means, but non-amino acid based linkers may also be used, according to principles and techniques also well known in the art and described in the literature. The linker may be cleavable (e.g. enzymatically) or non-cleavable.

[0068] GS polypeptides can be prepared as naked polypeptide chains or as modified polypeptides modified by binding or conjugation to additional moieties or chemical groups or substances. Exemplary modifications include, but are not limited to, pegylation, albumination, or other known modifications. For example, in some instances, GS polypeptides for use in the described methods are pegylated using standard methods well known in the art. This can, for example, help to increase the half-life of GS protein in circulation. Thus, in a preferred embodiment of the invention, GS protein may be provided as a conjugate with a polymer, such as polyethylene glycol (PEG) or a polysaccharide or oligosaccharide. Conjugates with PEG are particularly preferred. As indicated above, the preparation of such conjugates is well known in the art and described in the literature. Thus, PEGs of various sizes may be used to prepare the conjugates, for example from 100 Daltons to 100 kD, but more frequently from 5 kD to 100 kD, for example from 12 or 15 kD to 60 or 80 kD, for example from 15 to 50, 15 to 40, or 15 to 30 kD. Furthermore, PEGs may be bound or linked to the GS protein in various ways, and more than one PEG may be attached to each protein. PEGs may be linked directly or indirectly, for example via the linkers described above for the fusion proteins, or by any molecular or chemical group that may provide a linker function. Thus, PEGs may be linked at either or both of the N- or C-terminus, or internally to the GS molecule, for example at the amino groups of one or more lysine residues of the GS protein molecule, or at any other chemical moiety or residue of the protein molecule. Methods for binding or conjugating polymers such as PEG to proteins are well known in the art and described in the literature (see, for example, Roberts et al. 2012, Advanced Drug Delivery Reviews, 64 (supplement) 116-127 and Veronese 2001, Biomaterials 22, 405-417).The data presented in the following examples show that the PEG conjugates prepared by linking PEG to the N-terminus are particularly effective, for example in the activity assay of liver lysates from animals administered various conjugates.Therefore, the PEG conjugates comprising PEG linked to the N-terminus of GS protein are one preferred embodiment of the present invention.GS protein may be PEGylated in monomeric and / or multimeric form.Therefore, for convenience, preparations containing both monomeric and various multimeric forms of GS may be subjected to PEGylation.

[0069] GS can be formulated as a pharmaceutical composition for administration to a subject.GS can be formulated in any conventional manner by mixing a selected amount of GS with one or more physiologically or pharma-ceutically acceptable carriers or excipients.

[0070] Thus, a further aspect of the present invention provides a pharmaceutical composition comprising a GS protein and one or more pharma- ceutically acceptable carriers or excipients, for parenteral systemic administration.

[0071] The choice of carrier or excipient is within the skill of the administration specialist and may depend on several parameters, such as the mode of administration. In some examples, GS is provided as a liquid. In other examples, GS is provided in a dried form, such as a desiccated or lyophilized form. Such dried forms can be rehydrated before administration by adding a suitable solution, such as water, buffer, saline or other suitable solution. The GS provided herein may be formulated for direct administration or may be formulated for dilution or other modification. Thus, GS can be formulated in a single (or unit) dosage form or a multiple dosage form. Examples of single dosage forms include ampoules and syringes. Examples of multiple dosage forms include vials and bottles containing multiple unit doses.

[0072] The concentration of GS in the formulation is effective to deliver an amount of GS effective for converting ammonia to glutamine in the presence of glutamate when administered. The concentration and amount depend on several factors, including the substrate level in the subject and the mode of administration, and can be empirically determined. Exemplary concentrations of GS in the compositions provided herein include, but are not limited to, about 0.1, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000 or 5000 mg / mL GS or more.

[0073] To formulate a GS composition, in one embodiment, a weight fraction of GS is dissolved, suspended, dispersed, or otherwise mixed in a selected medium at a desired concentration. The resulting mixture can be formulated as a non-aqueous or aqueous mixture, including, but not limited to, a solution, suspension, paste, gel, aerosol, spray, or any other formulation suitable for systemic administration, such as a solution, suspension, emulsion, and other such mixtures.

[0074] Generally, GS compositions are prepared in consideration of approval from regulatory agencies or otherwise in accordance with generally recognized pharmacopoeias for use in animals and humans. GS compositions may include carriers such as diluents, excipients, or vehicles. Such pharmaceutical carriers may be sterile liquids such as water and oils. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. The compositions may contain, together with the active ingredient, diluents such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; lubricants such as magnesium stearate, calcium stearate, and talc; and binders such as starch, natural gums such as acacia, gelatin, glucose, molasses, polyvinylpyrrolidine, cellulose and its derivatives, povidone, crospovidone, and other such binders known to those skilled in the art. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, and ethanol. GS compositions can also contain, if desired, small amounts of wetting or emulsifying agents, or pH buffers, such as acetates, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharma-ceutically acceptable carriers.They can be prepared by methods known to those skilled in the art.Liposome delivery may also include sustained release formulations that include pharmaceutical matrices such as collagen gel and fibronectin-modified liposomes.

[0075] In addition to pharmaceutical compositions, GS can also be formulated or administered in other ways, for example, in nutritional compositions such as dietary supplements, for example parenteral nutritional compositions (for example, alone or together with other supplement components). GS can be included in such foods as a polypeptide (for example, a purified enzyme) or as part of an expression host cell or organism. Thus, for example, a microbial (for example, yeast or bacterial or fungal) host cell or a plant (including a plant cell) can be engineered to express GS and administered as such, for example, as a whole cell or an extract or other treatment (which can retain enzyme activity), or incorporated into a nutritional composition. Thus, for example, a bacterial or yeast cell suitable for human or non-human animal consumption can be engineered to express GS (i.e., by introduction of a nucleic acid molecule that includes a nucleotide sequence encoding GS). Alternatively, a plant can be engineered in an analagous manner, and suitable plant parts, etc. (for example, seeds, leaves, tubers, etc.) can be provided for administration. It is known in the art which microorganisms (e.g., yeast, bacteria, algae or fungi) are suitable for human or other animal consumption, and many such organisms are used today, for example, in probiotic preparations. Any such probiotic organism or preparation may be used, for example, based on lactic acid bacteria, such as Bifidobacterium or Lactobacillus species (e.g., Lactobacillus acidophilus). Thus, according to the present invention, such organisms or preparations may be formulated for the GI tract and may be administered directly to the GI tract, for example, by injection or infusion, or by enema or rectal administration. The exact amount or dose of GS administered to a subject depends on the activity of the GS, the route of administration, the disease or condition being treated, the number of doses, and other considerations, such as the weight, age and general condition of the subject. The particular dosage and administration protocol can be empirically determined or can be estimated, for example, from tests in animal models.Exemplary therapeutically effective doses of GS include, but are not limited to, from about 1 μg / kg to about 1000 μg / kg body weight per day, or from about 0.1 μg / kg body weight per day to about 10,000 μg / kg body weight per day, including from about 10 μg / kg to about 100 μg / kg body weight per day. Thus, for example, a subject may be administered 0.1, 0.2, 0.3, 0.4, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 400, 600, 800, 1000, 2000, 4000, 6000, 8000, 10,000, or 20,000 μg or more of GS per kg body weight per day.

[0076] It is a feature of the invention that GS is administered systemically but parenterally. "Oral" means by oral delivery. Thus, parenteral means that the GS protein is not administered by ingestion via the mouth. Other means of administration that deliver the GS protein to the intestine, or more generally to the gastrointestinal (GI) tract, may be included (e.g., rectally, or by enema, or direct administration to the GI tract), but are excluded in certain embodiments. Thus, in certain embodiments, the invention includes enteral administration, but in other embodiments does not. In further embodiments, the invention does not include administration to muscle, particularly skeletal muscle. Thus, in such embodiments, administration is not directed to muscle, i.e., non-muscle directed therapy.

[0077] Thus, GS can be administered by any method and route that delivers GS protein to the whole body, but does not include oral administration. In certain embodiments, GS protein can be administered parenterally. Those skilled in the art will be able to easily understand and select a suitable method of administration or delivery, including, but not limited to, intravenous, intramuscular, intradermal, transdermal, subcutaneous, or intraperitoneal administration, and any combination of any two or more thereof, designed in a manner suitable for each administration route. In some examples, the GS compositions described herein are administered subcutaneously. In other examples, the GS compositions are administered intravenously. For example, the GS compositions can be administered intravenously by injection or infusion, such as an intravenous bolus.

[0078] GS protein can also be administered in conjunction with or in combination with other therapeutic or active agents, in particular with second or further therapeutic agents that can treat (e.g. ameliorate) hyperammonemia. The second or further therapeutic agent that is active in treating hyperammonemia may alternatively be defined as an antihyperammonemia agent or an agent against hyperammonemia. The second or further agent can typically be a nitrogen scavenger (or ammonia scavenger) or an ammonia-reducing agent, such as a substituted amino acid or a urea cycle intermediate, or an analog thereof. Such agents can therefore include amino acids, such as arginine, glutamic acid, citrulline and / or ornithine, and / or N-acetylglutamic acid and / or the analog molecule carbamyl glutamic acid [Carbaglu®]. More suitably, the second or further agent is an ammonia-reducing agent, more suitably a nitrogen scavenger. Such an embodiment results in a particular aspect of the present invention.

[0079] The term "ammonia lowering agent" refers to a compound that removes and / or reduces ammonia or inhibits ammonia production. The ammonia lowering agent may be selected from the group consisting of nitrogen scavengers, ion exchange resins (e.g., Relapsa), ammonia absorbers (such as liposome-based ammonia absorbers, e.g., Versantis), engineered microbiomes that remove ammonia (e.g., Synlogic), rifaximin, and lactulose.

[0080] The term "nitrogen scavenger" as used herein refers to a compound that reduces the nitrogen and / or ammonia levels in a subject by removing ammonia.In a suitable embodiment, the nitrogen scavenger can be metabolized to phenylacetylglutamine and then excreted in urine, thereby reducing the amount of nitrogen and / or ammonia in a subject.

[0081] In suitable embodiments, the nitrogen scavenger may be selected from the group consisting of phenylacetic acid pharmaceutically acceptable salts (also referred to herein as phenylacetates), phenylbutyric acid pharmaceutically acceptable salts (also referred to herein as phenylbutyrates), glycerol phenylbutyrate, benzoic acid pharmaceutically acceptable prodrugs thereof, and ammonia binding resins. Other nitrogen scavengers will be known to those skilled in the art.

[0082] As used herein, the term "pharmaceutical acceptable salts" includes, for example, acid addition salts of phenylacetic acid that are sufficiently basic, such as, for example, acid addition salts with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, trifluoroacetic acid, formic acid, citric methane sulfonate or maleic acid. Furthermore, suitable pharmaceutical acceptable salts of phenylacetic acid that are sufficiently acidic are alkali metal salts, such as sodium salt or potassium salt, alkaline earth metal salts, such as calcium salt or magnesium salt, ammonium salt or salts with organic bases that provide pharmaceutical acceptable cations, such as, for example, salts with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.

[0083] In suitable embodiments, the pharma- ceutically acceptable salt of phenylacetic acid may be selected from the group consisting of sodium phenylacetate, potassium phenylacetate, ornithine phenylacetate.

[0084] In a suitable embodiment, the pharma- ceutically acceptable salt of phenylbutyric acid may be selected from the group consisting of sodium phenylbutyrate and potassium phenylacetate.

[0085] In suitable embodiments, the pharma- ceutically acceptable salt of benzoic acid may be selected from the group consisting of sodium benzoate and potassium benzoate.

[0086] It is to be understood that the ammonia reducing agents may exist in solvated and unsolvated forms, such as, for example, hydrated forms, and it is to be understood that encompassed within the context of the present invention are all such solvated and unsolvated forms.

[0087] The term "prodrug," as used herein, refers to an agent that has a chemical or biological moiety that renders it less active than an ammonia-lowering agent (such as a nitrogen scavenger), but that is metabolized to an ammonia-lowering agent or is hydrolyzed in vivo to form an ammonia-lowering agent.

[0088] In particular, agents that act to remove glutamine from the circulation (glutamine formed by the action of GS), such as phenylacetic acid or phenylbutyric acid compounds, are preferred. A second or further agent, such as an ammonia-lowering agent, may be administered separately, sequentially or simultaneously with the GS protein, including in the same formulation or composition, or in a separate composition or formulation.

[0089] Thus, in a further aspect, the present invention provides a product comprising a GS protein for systemic parenteral administration and a further therapeutic agent as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of hyperammonemia.

[0090] The second or additional agent may be administered by the same or a different route of administration, including orally. Thus, in one exemplary embodiment, the second or additional agent, such as an ammonia-lowering agent, may be administered orally or by other systemic means, and the GS may be administered by parenteral systemic means.

[0091] Ammonia-lowering agents such as nitrogen scavengers (including sodium phenylacetate, ornithine phenylacetate, sodium phenylbutyrate, or sodium benzoate) may be administered, for example, by intravenous infusion for acute management, and / or orally, for example, for long-term maintenance. The IV infusion may be peripheral, but central IV infusion is preferred. Similarly, amino acids such as arginine may be administered orally or by IV, for example central IV infusion.

[0092] Thus, a further aspect of the present invention provides a product (e.g., a combination product) that includes a GS protein for systemic parenteral administration and an additional therapeutic agent as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of hyperammonemia. In a suitable embodiment, such an additional therapeutic agent may be an ammonia-reducing agent. More suitably, the ammonia-reducing agent may be a nitrogen scavenger. More suitably, the nitrogen scavenger may be a pharma- ceutically acceptable salt of phenylacetic acid, more suitably sodium phenylacetate.

[0093] Alternatively, this aspect of the present invention may also be considered to provide a kit comprising (a) a GS protein for systemic parenteral administration and (b) a further therapeutic agent. Suitably, the further therapeutic agent may be effective against hyperammonemia. Suitably, the further therapeutic agent is an ammonia-reducing agent, more suitably a nitrogen scavenger.

[0094] Thus, in a further aspect, the present invention provides a kit comprising (a) a GS protein for systemic parenteral administration and (b) a nitrogen scavenger. Suitably, the nitrogen scavenger may be a pharma- ceutically acceptable salt of phenylacetic acid, more suitably sodium phenylacetate.

[0095] Such a kit may be provided for use in the treatment or prevention of hyperammonemia. The components of the kit may be provided as separate pharmaceutical compositions comprising the agent together with one or more pharma- ceutically acceptable carriers or excipients. The compositions of the invention may be administered once or more than once. If administered more than once, the compositions may be administered at regular intervals or as needed, e.g., as determined by a clinician. Regular intervals may include, for example, about daily, weekly, biweekly, monthly, or any other interval. The selection of a treatment protocol is well within the level of skill of the artisan. For example, the protocol may be determined based on testing in an animal model. In another example, repeated doses of the composition may be administered to the subject when the ammonia level in the blood exceeds a pre-determined level.

[0096] The use of the GS protein in combination with the GS protein or the use of an ammonia-reducing agent according to the present invention is advantageous in treating subjects for whom gene therapy is not appropriate or suitable, such as children, or subjects who are refractory to gene therapy. Such refractory subjects may include, for example, those who have been previously exposed to or have developed an immune response to the viral vector used to deliver the gene therapy.

[0097] Advantageously, the use of proteins as therapeutic agents allows for the delivery of higher doses of active protein to the subject and for the adjustment of the dose according to the subject and their needs. Moreover, compared to gene therapy, protein therapy allows for a much faster response and is therefore more suitable for emergency use.

[0098] As already mentioned, one aspect of the present invention relates to a pharmaceutical composition comprising an ammonia-lowering agent for use in combination with GS protein for use in the treatment or prevention of hyperammonemia. The ammonia-lowering agent can be formulated as a pharmaceutical composition for administration to a subject. The ammonia-lowering agent can be formulated in any conventional manner by mixing a selected amount of a nitrogen scavenger with one or more physiologically or pharma-ceutically acceptable carriers or excipients. Suitably, the composition may be for parenteral systemic administration or for oral administration. It will be understood that the pharmaceutical composition can also include GS protein. In such an embodiment, the composition will be formulated for parenteral systemic administration.

[0099] The choice of carrier or excipient is within the skill of the administration specialist and may depend on several parameters, such as the mode of administration. In some examples, the ammonia-lowering agent is provided as a liquid. In other examples, the ammonia-lowering agent is provided in a dry form. Such a dry form can be rehydrated prior to administration by addition of a suitable solution, such as water, buffer, saline or other suitable solution. The ammonia-lowering agent may be formulated for direct administration or may be formulated for dilution or other modification. Thus, the ammonia-lowering agent can be formulated in a single (or unit) dose form or a multiple dose form. Examples of single dose forms include ampoules and syringes. Examples of multiple dose forms include vials and bottles containing multiple unit doses.

[0100] The concentration of the ammonia-lowering agent in the formulation is effective to deliver an amount of the ammonia-lowering agent effective to remove nitrogen and / or ammonium from the circulation or reduce or inhibit ammonia production upon administration. The concentration and amount depend on several factors, including the substrate level in the subject and the mode of administration, and can be empirically determined. Exemplary concentrations of the ammonia-lowering agent in the compositions provided herein include, but are not limited to, about 0.1, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, or 5000 mg / mL ammonia-lowering agent or more.

[0101] To formulate the ammonia-lowering agent composition, in one embodiment, a weight fraction of the ammonia-lowering agent is dissolved, suspended, dispersed, or otherwise mixed in a selected medium at a desired concentration. The resulting mixture may be formulated as a non-aqueous or aqueous mixture, including, but not limited to, a solution, suspension, paste, gel, aerosol, spray, or any other formulation suitable for systemic administration, such as solutions, suspensions, emulsions, and other such mixtures.

[0102] Generally, ammonia lowering agents are prepared in consideration of approval from regulatory agencies or otherwise in accordance with generally recognized pharmacopoeias for animal and human use. The composition may include a carrier such as a diluent, excipient, or vehicle. Such pharmaceutical carriers may be sterile liquids such as water and oil. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. The composition may contain, together with the active ingredient, a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant such as magnesium stearate, calcium stearate, and talc; and a binder such as starch, natural gums such as acacia gum, gelatin, glucose, molasses, polyvinylpyrrolidine, cellulose and its derivatives, povidone, crospovidone, and other such binders known to those skilled in the art. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water and ethanol.Phenylacetic acid or its pharmaceutically acceptable salt composition can also contain a small amount of wetting agent or emulsifier, or pH buffer, such as acetate, sodium citrate, cyclodextrin derivative, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate and other such agents, if desired.Other examples of suitable pharmaceutical carriers will be known to those skilled in the art.

[0103] The ammonia-reducing agent can be administered by any method and route that delivers the compound to the body.In certain embodiments, the ammonia-reducing agent can be administered parenterally.Those skilled in the art will be able to easily understand and select the appropriate method of administration or delivery, including but not limited to intravenous, intramuscular, intradermal, transdermal, subcutaneous, or intraperitoneal administration, and any combination of any two or more thereof, formulated in a manner suitable for each administration route.

[0104] As well as pharmaceutical compositions, the ammonia-lowering agents may also be formulated or administered by other means, for example in nutritional compositions such as dietary supplements, for example parenteral nutritional compositions (e.g., alone or together with other supplement components). Suitably, such compositions may be for oral or parenteral administration.

[0105] As mentioned above, the ammonia-reducing agent is intended to be administered in combination with GS protein.It will be understood that the ammonia-reducing agent can be administered separately, sequentially or simultaneously with GS protein, including in the same formulation or composition, or in separate compositions or formulations.Thus, in one exemplary embodiment, the ammonia-reducing agent can be administered orally or by other systemic means, and GS can be administered by parenteral systemic means.

[0106] Exemplary therapeutically effective doses of an ammonia-lowering agent include, but are not limited to, about 10 mg / kg to about 1000 mg / kg body weight per day, or about 1 mg / kg to about 2000 mg / kg body weight per day, including about 100 mg / kg to about 500 mg / kg body weight per day. Thus, for example, a subject may be administered 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or 2000 mg or more of an ammonia-lowering agent per kg of body weight per day. Other exemplary therapeutically effective doses of the ammonia-lowering agent include, but are not limited to, about 1 g / day to about 50 g / day. Thus, for example, a subject may be administered 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 grams or more of the ammonia-lowering agent per day. It will be understood that the effective dose may vary depending on the ammonia-lowering agent. The GS composition, and / or the composition including the ammonia-lowering agent or the composition including an additional agent that is not an ammonia-lowering agent, may be presented in a package, kit, or dispenser device, such as a syringe with a needle, or a vial and a syringe with a needle, which may contain one or more unit dosage forms, if desired. The kit or dispenser device may be accompanied by instructions for administration. In embodiments where the GS composition and the composition including the ammonia-lowering agent are separate, the kit may include the GS composition and the composition including the ammonia-lowering agent. Suitably, in such embodiments, the kit may include the GS composition, and a nitrogen scavenger. Suitably, in such an embodiment, the kit may include a GS composition and a composition comprising a phenylacetate salt, such as sodium phenylacetate. The composition may be packaged as an article of manufacture containing packaging materials, the composition, and a label indicating that the composition is for administration to a subject to treat hyperammonemia or a disease or condition associated with hyperammonemia.

[0107] In a further aspect, the present invention provides an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof for use in the treatment or prevention of hyperammonemia, wherein the expression vector is for systemic administration.

[0108] In a suitable embodiment, the vector further encodes an ammonia-reducing agent.

[0109] In another aspect, the present invention provides an expression vector encoding glutamine synthetase, or a biologically active fragment or variant thereof, for use in combination with an ammonia-lowering agent for use in the treatment or prevention of hyperammonemia.

[0110] In another aspect, the present invention provides an ammonia-lowering agent for use in combination with an expression vector encoding glutamine synthetase, or a biologically active fragment or variant thereof, for use in the treatment or prevention of hyperammonemia.

[0111] In another aspect, the present invention provides a cell comprising an expression vector selected from the group consisting of an expression vector encoding glutamine synthetase, or a biologically active fragment or variant thereof, for use in the treatment of hyperammonemia, and an expression vector encoding glutamine synthetase, or a biologically active fragment or variant thereof, for use in combination with an ammonia-lowering agent, for use in the treatment or prevention of hyperammonemia.

[0112] In another aspect, the present invention provides the use of an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof for the manufacture of a composition for the treatment or prevention of hyperammonemia, wherein the composition is for systemic administration.

[0113] In another aspect, the present invention provides the use of an expression vector encoding glutamine synthetase, or a biologically active fragment or variant thereof, for the manufacture of a composition for use in combination with an ammonia-lowering agent for the treatment or prevention of hyperammonemia.

[0114] In another aspect, the present invention provides the use of an ammonia-lowering agent for the manufacture of a composition for use in combination with an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof, for use in the treatment or prevention of hyperammonemia.

[0115] In another aspect, the invention provides a method of treating or preventing hyperammonemia in a subject, the method comprising systemic administration of an expression vector encoding glutamine synthetase, or a biologically active fragment or variant thereof.

[0116] In another aspect, the invention provides a method of treating or preventing hyperammonemia in a subject, the method comprising administration of an expression vector encoding glutamine synthetase, or a biologically active fragment or variant thereof, and an ammonia-lowering agent.

[0117] In another aspect, the invention provides a composition comprising an expression vector encoding glutamine synthetase, or a biologically active fragment or variant thereof, for use in the treatment or prevention of hyperammonemia, wherein the composition is administered systemically.

[0118] In suitable embodiments, the composition may further comprise an ammonia reducing agent.

[0119] In another aspect, the present invention provides a composition comprising an expression vector encoding glutamine synthetase, or a biologically active fragment or variant thereof, for use in combination with an ammonia-lowering agent for use in the treatment or prevention of hyperammonemia.

[0120] In another aspect, the present invention provides a composition comprising an ammonia-lowering agent for use in combination with an expression vector encoding glutamine synthetase, or a biologically active fragment or variant thereof, for use in the treatment or prevention of hyperammonemia.

[0121] In another aspect, the present invention provides a kit comprising an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof, and a further therapeutic agent. Suitably, the further therapeutic agent may be an agent effective against hyperammonemia. Suitably, the agent effective against hyperammonemia is an ammonia-lowering agent or an amino acid or urea cycle intermediate thereof.

[0122] In another aspect, the present invention provides a product comprising an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof and a further therapeutic agent as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of hyperammonemia.Suitably, the further therapeutic agent may be an agent effective against hyperammonemia.Suitably, the agent effective against hyperammonemia is an ammonia-lowering agent or an amino acid or urea cycle intermediate thereof.More suitably, the further therapeutic agent is a nitrogen scavenger.

[0123] In suitable embodiments, the expression vector may be viral or non-viral. By way of example, suitable viral expression vectors may be derived from a virus selected from the group consisting of paramyxovirus, retrovirus, adenovirus, lentivirus, poxvirus, alphavirus, and herpesvirus. Other suitable viral vectors will be known to those skilled in the art.

[0124] Suitable non-viral expression vectors may be selected from the group consisting of inorganic particle expression vectors (such as calcium phosphate, silica, and gold), lipid-based particle expression vectors (e.g., cationic lipids, lipid nanoemulsions, and solid lipid nanoparticles), and polymer-based particle expression vectors (e.g., peptides, polyethyleneimines, chitosan, and dendrimers). Other suitable non-viral expression vectors will be known to those skilled in the art.

[0125] Suitable systemic administration methods will be known to those skilled in the art. By way of example, systemic administration may be achieved by parenteral administration routes, such as intravenous or subcutaneous routes. It will be understood that "systemic administration" allows the expression vector product (such as glutamine synthetase or a biologically active fragment thereof) to be expressed in multiple sites in a patient. In the context of the present invention, systemic administration does not include intramuscular administration.

[0126] The term "biologically active" refers to a fragment or variant of the nucleic acid encoding glutamine synthetase (SEQ ID NO: 1) that exhibits the ability to convert glutamate to glutamine. The protein set forth in SEQ ID NO: 1 is encoded by the nucleic acid sequence set forth in SEQ ID NO: 2. Thus, it will be understood that a vector can include a fragment or variant of SEQ ID NO: 2 that encodes a biologically active fragment or variant of glutamine synthetase.

[0127] The term "variant" as used herein refers to a polypeptide that includes a modification of the primary structure of the polypeptide of SEQ ID NO:1. Suitably, a variant may share 70% or more identity with the polypeptide of SEQ ID NO:1; 80% or more identity with the polypeptide of SEQ ID NO:1; 90% or more identity with the polypeptide of SEQ ID NO:1; 95% or more identity with the polypeptide of SEQ ID NO:1; 96% or more identity with the polypeptide of SEQ ID NO:1; 97% or more identity with the polypeptide of SEQ ID NO:1; 98% or more identity with the polypeptide of SEQ ID NO:1; or even 99% or more identity with the polypeptide of SEQ ID NO:1. A variant may differ from the polypeptide of SEQ ID NO:1 by 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more, 20% or more, or even 30% or more with respect to the sequence set forth in SEQ ID NO:1.

[0128] The term "fragment" as used herein refers to a polypeptide that comprises a modification of the length of the primary structure of the polypeptide of SEQ ID NO: 1. Suitable fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the full length of SEQ ID NO: 1. Indeed, suitable variants may comprise at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 1.

[0129] It will be understood that (unless the context requires otherwise) the embodiments described with respect to GS proteins for use in the prevention of hyperammonemia, GS proteins for use in combination with ammonia-lowering agents, ammonia-lowering agents for use in combination with GS proteins, their uses, methods of treatment, compositions, kits and products are generally applicable to the remaining aspects of the invention.

[0130] The invention will now be further described with reference to the following non-limiting examples and figures. The present disclosure relates, for example, to the following: [Section 1] A glutamine synthetase (GS) protein for use in the treatment or prevention of hyperammonemia by systemic parenteral administration to a subject. [Section 2] Item 2. The protein for use according to item 1, wherein the protein is for use in combination with an ammonia-lowering agent. [Section 3] An ammonia-reducing agent for use in combination with a GS protein for use in the treatment or prevention of hyperammonemia. [Section 4] The protein or ammonia-reducing agent for use according to any one of items 1 to 3, wherein the hyperammonemia is caused by urea cycle disorder (UCD) and / or glutamine synthetase deficiency. [Section 5] Item 5. The protein or ammonia-reducing agent for use according to any one of items 1 to 4, wherein hyperammonemia is associated with organ failure. [Section 6] Item 6. The protein or ammonia-reducing agent for use according to any one of items 1 to 5, wherein hyperammonemia is caused by non-alcoholic fatty liver disease. [Section 7] The protein or ammonia-reducing agent for use according to any one of items 1 to 5, wherein the hyperammonemia is caused by acute liver failure, liver cirrhosis, and / or renal dysfunction and / or renal failure. [Section 8] A protein or ammonia lowering agent for use according to any of the preceding claims, wherein the GS protein comprises an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO:1, or is an enzymatically active fragment thereof. [Section 9] The protein or ammonia-lowering agent for use according to any one of the preceding paragraphs, wherein the GS protein is administered in the form of a pharmaceutical composition. [Section 10] The protein or ammonia-lowering agent for use according to any of the preceding paragraphs, wherein the GS protein is administered in the form of a parenteral nutrition composition. [Section 11] 4. A protein or an ammonia-lowering agent for use according to any of the preceding claims, wherein the GS protein is linked to a moiety. [Section 12] 7. A protein or ammonia-reducing agent for use according to any of the preceding clauses, wherein the moiety is selected from a protein, a peptide, a non-proteinaceous polymer or an affinity tag. [Section 13] 7. The protein or ammonia-reducing agent for use according to any of the preceding claims, wherein the moiety is polyethylene glycol (PEG). [Section 14] Item 2. A protein or ammonia-lowering agent for use according to any of the preceding paragraphs, wherein the PEG is linked to the GS protein at the N-terminus of the protein. [Section 15] 4. The protein or ammonia-lowering agent for use according to any of the preceding claims, wherein the GS protein is linked to the moiety via a peptide linker or chemical bond. [Section 16] 13. The protein or ammonia lowering agent for use according to any one of claims 8 to 12, wherein the GS protein is linked to the moiety via a covalent bond. [Section 17] The protein or ammonia-lowering agent for use according to any of the preceding paragraphs, wherein the GS protein is in a form suitable for parenteral administration to a subject. [Section 18] The protein or ammonia-lowering agent for use according to any of the preceding paragraphs, wherein the GS protein is in a form suitable for subcutaneous administration to a subject. [Section 19] Item 2. The protein or ammonia-lowering agent for use according to any of the preceding paragraphs, wherein the GS protein is provided as a preparation comprising a multimeric form of the protein. [Section 20] Item 2. The protein or ammonia-lowering agent for use according to any of the preceding paragraphs, wherein the GS protein is provided in monomeric form. [Section 21] 21. The protein or ammonia-lowering agent for use according to any one of claims 2 to 20, wherein the ammonia-lowering agent is selected from the group consisting of nitrogen scavengers, ion exchange resins (e.g., Relapsa), ammonia absorbers (such as liposome-based ammonia absorbers, e.g., Versantis), engineered microbiomes that remove ammonia (e.g., Synlogic), rifaximin and lactulose. [Section 22] Item 22. The protein or nitrogen scavenger for use according to item 21, wherein the nitrogen scavenger is selected from the group consisting of a pharma- ceutically acceptable salt of phenylacetic acid or a pharma- ceutically acceptable prodrug thereof, a pharma- ceutically acceptable salt of phenylbutyric acid or a pharma- ceutically acceptable prodrug thereof, glycerol phenylbutyrate or a pharma- ceutically acceptable prodrug thereof, a pharma- ceutically acceptable salt of benzoic acid or a pharma- ceutically acceptable prodrug thereof, and an ammonia binding resin. [Section 23] Item 23. The protein or nitrogen scavenger for use according to Item 22, wherein the pharma- ceutically acceptable salt of phenylacetic acid is sodium phenylacetate. [Section 24] Use of a GS protein for the manufacture of a composition for the treatment or prevention of hyperammonemia by systemic parenteral administration to a subject. [Section 25] Use of an ammonia-lowering agent for the manufacture of a composition for use in combination with a GS protein for the treatment or prevention of hyperammonemia. [Section 26] Use of a GS protein and an ammonia-reducing agent for the manufacture of a composition for use in the treatment or prevention of hyperammonemia. [Section 27] The use of a GS protein and / or an ammonia-reducing agent according to clause 24 or 26, wherein hyperammonemia is as defined in clauses 4 to 7. [Section 28] Use of a GS protein and / or an ammonia-lowering agent according to any one of clauses 24 to 27, wherein the GS protein is as defined in clauses 8 to 20. [Section 29] 29. The use of a GS protein and / or ammonia-lowering agent according to any one of claims 24 to 28, wherein the ammonia-lowering agent is selected from the group consisting of nitrogen scavengers, ion exchange resins (e.g., Relapsa), ammonia absorbers (such as liposome-based ammonia absorbers, e.g., Versantis), engineered microbiomes that remove ammonia (e.g., Synlogic), rifaximin and lactulose. [Section 30] 30. Use of a GS protein and / or an ammonia-lowering agent according to any one of claims 24 to 29, wherein the nitrogen scavenger is as defined in claim 22 or 23. [Section 31] A method for treating or preventing hyperammonemia in a subject, comprising systemically and parenterally administering to said subject a GS protein. [Section 32] 32. The method of claim 31, further comprising administering an ammonia-lowering agent. [Section 33] 33. The method of clause 31 or 32, wherein the hyperammonemia is as defined in clauses 4 to 7. [Section 34] The method of any one of claims 31 to 33, wherein the GS protein is as defined in any one of claims 8 to 20. [Section 35] 35. The method of claim 32 to 34, wherein the ammonia-reducing agent is selected from the group consisting of nitrogen scavengers, ion exchange resins (e.g., Relapsa), ammonia absorbers (such as liposome-based ammonia absorbers, e.g., Versantis), engineered microbiomes that remove ammonia (e.g., Synlogic), rifaximin, and lactulose. [Section 36] 36. The method of claim 35, wherein the nitrogen scavenger is as defined in claim 22 or 23. [Section 37] A composition comprising a GS protein for use in the treatment or prevention of hyperammonemia, wherein the composition is in a form suitable for systemic parenteral administration. [Section 38] A composition comprising an ammonia-lowering agent for use in combination with a GS protein for use in the treatment or prevention of hyperammonemia. [Section 39] A composition comprising a GS protein and an ammonia-lowering agent. [Section 40] Item 40. The composition according to item 39, wherein the composition is for use in the treatment or prevention of hyperammonemia. [Section 41] 41. The composition of any one of claims 37 to 40, wherein the composition is a pharmaceutical composition or a nutritional composition. [Section 42] 42. The composition of any one of claims 37 to 41, wherein the composition comprises at least one pharma- ceutically acceptable carrier or excipient. [Section 43] 43. The composition of any one of paragraphs 38 to 42, wherein the composition is in a form suitable for systemic parenteral administration. [Section 44] 44. The composition of any one of paragraphs 37 to 43, wherein the composition is in a form suitable for subcutaneous administration. [Section 45] 45. The composition of any one of paragraphs 37 to 44, wherein the composition comprises a further therapeutic agent. [Section 46] 46. ​​The composition of any one of clauses 37 to 45, wherein the hyperammonemia is as defined in clauses 4 to 7. [Section 47] 47. The composition of any one of clauses 37 to 46, wherein the GS protein is as defined in clauses 8 to 20. [Section 48] 48. The composition of any one of clauses 38 to 47, wherein the ammonia-reducing agent is selected from the group consisting of nitrogen scavengers, ion exchange resins (e.g., Relapsa), ammonia absorbers (such as liposome-based ammonia absorbers, e.g., Versantis), engineered microbiomes that remove ammonia (e.g., Synlogic), rifaximin, and lactulose. [Section 49] 49. The composition according to claim 48, wherein the nitrogen scavenger is as defined in claim 22 or 23. [Section 50] A kit comprising a GS protein and an additional therapeutic agent for systemic parenteral administration. [Section 51] 51. The kit of paragraph 50, wherein the additional therapeutic agent is effective against hyperammonemia. [Section 52] 52. The kit of paragraph 50 or 51, wherein the additional therapeutic agent is an ammonia lowering agent or an amino acid or urea cycle intermediate or analog thereof. [Section 53] 53. The kit of claim 52, wherein the ammonia-reducing agent is selected from the group consisting of nitrogen scavengers, ion exchange resins (e.g., Relapsa), ammonia absorbers (such as liposome-based ammonia absorbers, e.g., Versantis), engineered microbiomes that remove ammonia (e.g., Synlogic), rifaximin, and lactulose. [Section 54] 54. The kit of claim 53, wherein the nitrogen scavenger is as defined in claim 22 or 23. [Section 55] 55. The kit of any one of clauses 50 to 54, wherein the hyperammonemia is as defined in clauses 4 to 7. [Section 56] 56. The kit of any one of paragraphs 50 to 55, wherein the GS protein is as defined in paragraphs 8 to 20. [Section 57] A product comprising a GS protein and a further therapeutic agent for systemic parenteral administration as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of hyperammonemia. [Section 58] 58. The product of paragraph 57, wherein the further therapeutic agent is an ammonia lowering agent or an amino acid or a urea cycle intermediate or analogue thereof. [Section 59] 59. The product according to clause 58, wherein the ammonia-reducing agent is selected from the group consisting of nitrogen scavengers, ion exchange resins (e.g. Relapsa), ammonia absorbers (such as liposome-based ammonia absorbers, e.g. Versantis), engineered microbiomes that remove ammonia (e.g. Synlogic), rifaximin and lactulose. [Section 60] 60. The product of claim 59, wherein the nitrogen scavenger is as defined in claim 22 or 23. [Section 61] 61. The product of any one of clauses 57 to 60, wherein hyperammonemia is as defined in clauses 4 to 7. [Section 62] 62. The product of any one of clauses 57 to 61, wherein the GS protein is as defined in clauses 8 to 20. [Section 63] 1. An expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof for use in the treatment or prevention of hyperammonemia, wherein the expression vector is for systemic administration. [Section 64] 64. An expression vector for use according to paragraph 63, further encoding an ammonia reducing agent. [Section 65] 1. An expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof for use in combination with an ammonia-lowering agent for use in the treatment or prevention of hyperammonemia. [Section 66] 13. An ammonia-reducing agent for use in combination with an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof for use in the treatment or prevention of hyperammonemia. [Section 67] A cell comprising an expression vector for use as defined in paragraphs 63 to 66. [Section 68] 1. Use of an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof for the manufacture of a composition for the treatment or prevention of hyperammonemia, wherein the composition is for systemic administration. [Section 69] 2. Use of an expression vector encoding glutamine synthetase, or a biologically active fragment or variant thereof, for the manufacture of a composition for use in combination with an ammonia-lowering agent for the treatment or prevention of hyperammonemia. [Section 70] 13. Use of an ammonia-reducing agent for the manufacture of a composition for use in combination with an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof for use in the treatment or prevention of hyperammonemia. [Section 71] SUMMARY OF THE DISCLOSURE A method for treating or preventing hyperammonemia in a subject comprising systemic administration of an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof. [Section 72] A method for treating or preventing hyperammonemia in a subject, the method comprising administration of an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof, and an ammonia-lowering agent. [Section 73] 1. A composition comprising an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof for use in the treatment or prevention of hyperammonemia, the composition being for systemic administration. [Section 74] 74. The composition of claim 73, further comprising an ammonia reducing agent. [Section 75] A composition comprising an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof for use in combination with an ammonia-lowering agent for use in the treatment or prevention of hyperammonemia. [Section 76] A composition comprising an ammonia-lowering agent, for use in combination with an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof, for use in the treatment or prevention of hyperammonemia. [Section 77] A kit comprising an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof, and an additional therapeutic agent. [Section 78] 78. The kit of paragraph 77, wherein the additional therapeutic agent may be an agent effective against hyperammonemia. [Section 79] 79. The kit of claim 78, wherein the agent effective against hyperammonemia is an ammonia-lowering agent or an amino acid or urea cycle intermediate thereof. [Section 80] A product comprising an expression vector encoding glutamine synthetase or a biologically active fragment or variant thereof and a further therapeutic agent as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of hyperammonemia. [Section 81] The product of clause 80, wherein the further therapeutic agent is as defined in clause 78 or 79. [Brief description of the drawings]

[0131] [Figure 1] 1 shows the results of size exclusion chromatography (SEC) on a Superose 12 column of the 20 kDa N-terminal aldehyde PEG conjugate of human GS protein prepared in Example 1. The graph shows that multimers were eluted in fractions 8 and 9, and monomers in fraction 10. [Diagram 2] Figure 2 shows a comparison of in-vitro GS activity of various GS candidates: PEG-conjugated mutants (Trin GS1, Trin GS2, Trin GS3, Trin GS 4) versus non-conjugated GS (wt GS) and negative control. Glutamine synthetase activity is shown as OD570nm according to the assay of Acosta et al., 2009, World J. Gastroenterol.,15(23), 2893-2899. Trin GS1 - (N-terminal aldehyde monomer);Trin GS2 - Nof-20;Trin GS3 - Nof-30, Trin GS4 - N-terminal aldehyde monomer. [Diagram 3] FIG. 3 shows PEG ELISA results for pre- and post-dose plasma of male, wild-type (wt) CD1 mice at (A) baseline, (B) 24 hours post-dose, and (C) 72 hours post-dose for various conjugates (Trin1 - N-terminal aldehyde conjugated GS monomer; Trin2 - Nof-20 GS conjugated multimer; Trin3 - Nof-30 conjugated GS multimer; Trin4 - N-terminal aldehyde conjugated PEG multimer). [Figure 4]FIG. 4 shows the results of GS activity (OD535nm) in liver lysates in wt CD1 mice treated with 2.5 mg / kg, 3 days after treatment, as described in Example 3. [Diagram 5] Figure 5A shows the results of a liver GS activity assay, and Figure 5B shows the results of a plasma GS activity assay. Both liver and plasma GS activities were assayed in BDL rats treated with GS protein, and GS protein and a nitrogen scavenger. [Figure 6] FIG. 6 illustrates ammonia blood levels measured in BDL rats treated with GS protein and GS protein plus a nitrogen scavenger. [Figure 7] FIG. 7 illustrates a graph showing the percentage of prefrontal cortex edema in BDL rats treated with GS protein or GS protein and a nitrogen scavenger. [Figure 8] FIG. 8 shows the results of the rotarod grip strength test of BDL rats treated with GS protein or GS protein and a nitrogen scavenger. [Figure 9] FIG. 9 is a graph showing ammonia levels in OTC mice treated with GS protein or GS protein plus a nitrogen scavenger (SP - sodium phenylacetate). [Figure 10] FIG. 10 shows the results of ammonia levels in plasma and hepatic GS activity in OTC mice treated with GS protein or DS protein and a nitrogen scavenger (SP - sodium phenylacetate). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0132] Preparation and purification of GS proteins and GS protein-PEG conjugates Construction of human glutamine synthetase (GS): pET30a containing the gene for human GS (sequence number 5, codon-optimized for expression in bacteria, including a His-tag and 5' sequence encoding the linker GGGGS at the N-terminus of GS) +The vector was used in an Escherichia coli expression system. After plasmid construction, evaluation of GS expression was performed at a wide range of induction (IPTG) and expression temperatures. Human GS was soluble expressed in the construct detected by SDS-PAGE. Soluble protein was extracted from the cells using lysis buffer (50 mM Tris pH 8.0, 10% glycerol, 0.1% Triton X-100, 100ug / ml lysozyme, 1mM PMSF, 3 units of DNAse, 2mM MgCl). Soluble protein was extracted after centrifugation. After expression testing, the best condition was found in BL21(DE3) cells, which were cultured and induced with 0.1mM IPTG at 25°C for 16 hours. Other conditions tried included the use of diverse IPTG inductions (0.01M to 0.1M IPTG), various incubation temperatures (ranging from 16°C to 37°C), and induction incubation times from 4 to 16 hours.

[0133] Purification of expressed GS: The initial purification steps of the expressed protein involved His-tag purification with Ni-NTA beads, washing with 20 mM imidazole, and elution with 300 mM imidazole.

[0134] Protein PEG conjugation: GS proteins were conjugated to N-terminal aldehyde 20 kDa PEG under reducing conditions (by using 20 mM sodium cyanoborohydride) for 16 hours (Dr Reddy's 20 kDa N-terminal Aldehyde PEG).

[0135] Final purification: The conjugated proteins were further purified using SEC chromatography. Superose 6 or Superose 12 columns (see Figure 1) were used. Multimers were found in fractions 8 ± 9. Fraction 10 of Superose 12 contained (low concentrations) multimers. On Superose 6, multimers were found in fractions 8 + 9 and monomers in fractions 12 / 13.

[0136] The final formulation of GS was prepared in PBS, pH 7.4, containing trehalose and sucrose. EXAMPLES

[0137] Activity of GS preparations Various GS preparations and PEG conjugates prepared according to Example 1 were tested for GS activity using the assay of Acosta et al., 2009 (see above), which was modified from the original assay described in Ehrenfeld et al., 1963, J. Biol. Chem. 238(11), 3711-3716.

[0138] 100ug of purified protein sample was added to the following reaction buffer: 150μL stock solution (100mmol / L imidazole-HCl buffer [pH 7.1], 40mmol / L MgCl2, 50mmol / L β-mercaptoethanol, 20mmol / L ATP, 100mmol / L glutamic acid, and 200mmol / L hydroxylamine adjusted to pH 7.2). The tube was incubated at 37°C for 15 minutes. The reaction was stopped by adding 0.6mL [2x concentration] ferric chloride reagent (0.37mol / L FeCl3, 0.67mol / L HCl, and 0.20mol / L trichloroacetic acid). The sample was placed on ice for 5 minutes. Precipitated protein was removed by centrifugation at 10,000g, and the absorbance of the supernatant was read at 535-570nm against a reagent blank. The results are shown in Figure 2. Trin1 - (20kD size N-terminal aldehyde monomeric PEG obtained from Dr Reddy's); Trin2 - Nof-20 conjugated GS, monofunctional linear 20kD PEG, NHS active ester and conjugated to GS protein obtained from NOF corporation; Trin3 - Nof-30, monofunctional linear 30kD PEG, NHS active ester and conjugated to GS protein obtained from NOF corporation; Trin4 - N-terminal aldehyde, GS multimer). The activities of the other conjugates were similar, but Trin4 (N-terminal aldehyde multimer) showed the best activity and had a very similar activity profile compared to wt GS (unconjugated). EXAMPLES

[0139] Administration of GS protein to mice - effect on plasma levels of GS protein-PEG conjugate Male wild type (wt) CD1 mice were dosed at 2.5 mg / kg by subcutaneous (sc) administration with various GS proteins and PEG conjugates (Trin1 - N-terminal aldehyde conjugated GS monomer; Trin2 - Nof-20 GS conjugated multimer; Trin3 - Nof-30 conjugated GS multimer; Trin4 - N-terminal aldehyde conjugated PEG multimer) prepared as described in Example 1. ELISA was performed according to the protocol outlined by the manufacturer (Abcam PEG ELISA kit, ab133065). Plasma ELISA results shown in Figure 3 show, as expected, extremely low or undetectable levels for unconjugated wt GS at all time points. After 24 hours, several candidates were found to be at high levels in plasma, but after 72 hours post-dose, Trin-GS 4 (N-terminal aldehyde conjugated PEG GS multimer) showed the highest presence. N=2 per group. Thus, this experiment demonstrates that systemic administration of GS proteins can be used successfully to obtain high circulating levels of GS PEG conjugates, and in particular levels that may be therapeutically effective or active. EXAMPLES

[0140] Administration of GS protein to mice – GS activity levels in liver lysates Activity assays were performed as described in Example 2, except that 500 μg of liver lysate (from mice selected from the experiment in Example 3) was added to each reaction as appropriate. Results are shown in Figure 4. GS activity results in liver lysates 3 days post-treatment indicate that the superior candidate was the N-terminal aldehyde conjugated PEG GS multimer, the only candidate showing significant activity above baseline compared to vehicle (saline-treated) control. N=2 per group. EXAMPLES

[0141] Urea cycle disorder (OTC deficiency) spf-ash A mouse model was used to demonstrate the effects of GS and GS+SP. Details of the mice used can be found at https: / / www.jax.org / strain / 001811(B6EiC3Sn a / A-Otc spf-ash Mice were fed a normal diet. Ages ranged from approximately 10 to 23 weeks and groups were well matched. All animals were male hemizygous (OTC is X-linked, so it is only present on the X chromosome in males, hence the mice are knockouts).

[0142] All groups [vehicle, GS and GS+SP; GS=Glutamine Synthetase, GS+SP=Glutamine Synthetase+Sodium Phenlyacetate] were treated as follows: The experiment was conducted from Tuesday to Wednesday of the following week (8 days). SP was administered 350 mg / kg ip twice daily; GS was administered to all treatment groups for the first 4 days (40 mg / kg ip once daily), then discontinued for 2 days [over the weekend] and GS was administered at 40 mg / kg ip for an additional 3 days.

[0143] Mice were culled on day 8, blood was extracted and spun down for plasma, which was used for ammonia quantification (see methods below).

[0144] Genotyping is performed using standard methods described in the literature.

[0145] material and method All experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986, as amended in accordance with European Directive 2010 / 63 / EU. All animals received humane care according to the standards outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication 86-23; revised 1985). All animals used in these experiments were male Sprague-Dawley rats (weight at the start of the experiment, 250 g) obtained from Charles River Laboratories (Kent, UK) and divided into five groups: bile duct ligated animals + ammonia + saline serum (BDL+HA+SS, n=6), bile duct ligated animals + ammonia + sodium phenylacetate (BDL+HA+SP, n=6), bile duct ligated animals + ammonia + sodium phenylacetate + glutamine synthetase (BDL+HA+SP+GS, n=5), bile duct ligated animals + ammonia + glutamine synthetase (BDL+HA+GS, n=6), and sham-operated animals + glutamine synthetase (SHAM+GS, n=5). Treatments including SP and GS are sometimes referred to as "COMBO".

[0146] Bile duct ligation surgery Under general anesthesia (5% isoflurane in 100% oxygen for induction, 2% isoflurane in air for maintenance), rats underwent triple ligation of the bile duct (minilaparotomy approach) to induce chronic liver injury and were examined 28 days after surgery. A midline laparotomy was performed under anesthesia. In the BDL group, the common bile duct was isolated, triple ligated with 3-0 silk thread, and incised between the ligated sites. The sham-operated group underwent the same procedure without incision between the ligated sites. After BDL, all animals continued to gain weight and were comparable to sham-operated controls. Overall mortality in both groups was less than 10% and occurred within 36 hours of surgery.

[0147] Non-cirrhotic hyperammonemic conditions Twenty-three rats were fed a hyperammonemic (HA) diet. The amino acid recipe used for approximately 100 g of stock was 15 g leucine, 7.7 g phenylalanine, 7 g glutamic acid, 10 g alanine, 4.4 g proline, 5.8 g threonine, 11 g aspartic acid, 5 g serine, 4.8 g glycine, 3.3 g arginine, 9.6 g lysine, 8.4 g histidine, 3 g tyrosine, 1.5 g tryptophan, and 10.6 g valine. 25 g of this mix (mixed 1:5 with standard rodent chow powder) was freshly prepared each day, and the rats had free access to it for 5 days. The recipe approximated the amino acid composition of rodent hemoglobin, mimicking the effects of gastrointestinal bleeding, which is known to result in systemic hyperammonemia [2], [1].

[0148] Sodium Phenylacetate Conditions Eleven rats were fed a sodium phenylacetate (SP) diet, 0.3 g / kg per day, for 5 days, mixed with diet powder and prepared fresh each day.

[0149] Glutamine synthetase conditions Sixteen rats were injected intraperitoneally with GS once every two days (days 1 and 3). The total volume injected was 3 ml ip allowing for 18-22 mg / kg of GS.

[0150] Blood sampling and biochemistry Plasma samples were taken from a leg vein in all groups at different time points. The time points after treatment with glutamine synthetase were counted as follows: 6 h, 24 h, 48 h and 5 days. Analysis of plasma ammonia levels at each time point was performed using 200 μl of plasma using a Cobas Integra 400 multianalyser (Roche-diagnostics, Burgess Hill, West Sussex, UK) equipped with the appropriate kit.

[0151] Cerebral edema This was measured using the dry weight method as described previously [3, 4]. Briefly, oven-dried Eppendorfs were weighed on a sensitive electronic balance, then the prefrontal cortex, striatum, hippocampus, cerebellum and cortex of each animal were placed into their respective labeled Eppendorfs and reweighed; all samples, within 0.1 mg. Dry weights were determined after drying Eppendorfs filled with individual brain samples in an oven at 60 °C for 7 days. Tissue moisture content was then calculated as %H2O = (1 - dry wt / wet wt) × 100%.

[0152] Tests for assessing locomotor activity: rotarod-accelerod test This test of motor skills consists of a motorized rotating rod that allows us to evaluate motor coordination and fatigue resistance (Jones and Roberts 1968). An accelerating rotarod 7750 from Ugo Basile (Ugo Basile Biological Research Apparatus, Italy) was used for the rats. The procedure that followed had two parts. In the first part, the animals were placed in the apparatus and the speed was kept constant at 2 rpm for 60 seconds. In the second part, the rats were evaluated for 5 minutes in an accelerating rod test session in which the rotation speed was constantly increased until it reached 20 rpm. The time taken to fall off the rod and the actual rotation speed were recorded in pre- and post-treatment conditions for all groups after 1 hour of treatment.

[0153] Determination of ammonia in blood using the TCA direct method Plasma ammonia concentrations were measured using the method described in the paper (Clin Chim Acta. 1968 Oct;22(2)183-86) as follows.

[0154] principle In alkaline solution, ammonium ions react with hypochlorite to form monochloramine. In the presence of phenol and excess hypochlorite, and using nitroprusside as a catalyst, monochloramine forms the blue compound, indophenol. The concentration of ammonium is determined spectrophotometrically at 630 nm.

[0155] method Prepare reagent A by dissolving 3.5 g of phenol and 0.04 g of sodium nitroprusside in 100 ml of distilled water. Prepare Reagent B by dissolving 1.8 g sodium hydroxide in 48 ml distilled water and adding to 4 ml of 1 M sodium hypochlorite solution. 150 μl of 5% TCA is added to 50 μl of each plasma sample and centrifuged at 10,000 RPM for 10 minutes at 4° C. 50 μl of the supernatant is taken and placed in a 96-well plate and 50 μl of both Reagents A and B are added. Prepare standard ammonium chloride concentrations for the calibration curve by dissolving ammonium chloride in distilled water and serially diluting to give concentrations from 400 μmol to 3 μmol. Use distilled water as a blank. The well plate is protected from light and incubated for 60 min at 50° C. The ammonia concentration is determined by measuring the absorbance at 630 nm using a spectrophotometer.

[0156] result Administration of GS protein to mice – GS activity levels in liver and blood Activity assays were performed as described in the Materials and Methods section above. The results are shown in Figures 5A and B. The results of rat liver measured on day 5 show that the SHAM+GS group had the best GS activity. Furthermore, it can be seen from Figure 5A that GS and GS+SP treatments increase GS activity in the liver of mice receiving BDL. When measured in blood, the results show that the BDL+GS group had the best GS activity. Furthermore, it can be seen from Figure 5B that GS activity in blood is consistent over time, even at 24 and 48 hours after administration.

[0157] Administration of GS protein to mice - Ammonia concentration in BDL rats As seen in Figure 6, ammonia levels are highest in BDL rats. GS, GS+SP, and SP treatments each resulted in a significant reduction in ammonia levels in the blood. GS reduced ammonia levels after two doses. GS+SP treatment reduced ammonia levels most significantly, suggesting a synergistic effect.

[0158] Administration of GS protein to mice - Brain swelling in BDL rats Brain edema was measured in the prefrontal cortex. GS treatment was found to most significantly reduce brain edema compared to SP treatment, and even SP+GC treatment (Figure 7). SP treatment did not statistically significantly reduce swelling compared to controls (i.e., BDL mice without treatment).

[0159] Administration of GS protein to mice - Brain and physical functions in BDL rats Figure 8 shows the results of the rotarod grip strength test. Surprisingly, GS administration was found to improve performance in all tested mouse groups. SP treatment alone did not lead to a statistically significant effect, while GS+SP treatment showed the best improvement, suggesting a synergisic effect.

[0160] Treatment of mice with OTC deficiency As shown in FIG. 9, ammonia is highly significantly reduced in the treatment groups.

[0161] In Figure 10, it can be seen that plasma GS activity increased in OTC mice treated with GS or GS&SP from 0.2 in the vehicle group to 0.8 in the GS only group and about 1.1 in the GS&SP group. Liver GS activity increased from about 0.175 in the vehicle group to about 2.8 in the GS only group and about 2.5 in the GS&SP group.

[0162] Summary of results In summary, administered GS is biocompatible and safe, and improves blood and liver GS activity, which also leads to a reduction in ammonia and cerebral edema, as well as improved neurocognitive and / or physical function. Furthermore, the data suggest that SP and GS treatment may have a synergistic effect. (References) [1] Riggs A. The amino acid composition of some mammalian hemoglobins: mouse, guinea pig, and elephant. J Biol Chem 1963;238:2983-2987. [2] Balata S, Olde Damink SW, Ferguson K, Marshall I, Hayes PC, Deutz NE, Williams R, Wardlaw J, Jalan R. Induced hyperammonemia alters neuropsychology, brain MR spectroscopy and magnetization transfer in cirrhosis. Hepatology 2003;37:931-939. [3] Stewart-Wallace AM. A biochemical study of cerebral tissue, and of changes in cerebral oedema. Brain 1939; 62: 426-38. [4] Traber PG, Ganger DR, Blei AT. Brain edema in rabbits with galactosamine-induced fulminant hepatitis. Regional differences and effects on intracranial pressure. Gastroenterology 1986; 91: 1347-56. array: SEQ ID NO:1 [fully human protein] MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRACLYA GVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN Sequence 2 (only methionine is cleaved for the mature protein in vivo): TTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRACLYAG VKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN SEQ ID NO:3 cDNA CGAGAGTGGGAGAAGAGCGGAGCGTGTGAGCAGTACTGCGGCCTCCTCTCCTCTCCTAAC CTGCTCTCGCGGCCTACCTTTACCCGCCGCCTGCTCGGCGACCAGAACACCTTCCACCA TGACCACCTCAGCAAGTTCCCACTTAAATAAAGGCATCAAGCAGGTGTACATGTCCCTGC CTCAGGGTGAGAAAGTCCAGGCCATGTATATCTGGATCGATGGTACTGGAGAAGGACTGC GCTGCAAGACCCGGACCCTGGACAGTGAGCCCAAGTGTGTGGAAGAGTTGCCTGAGTGGA ATTTCGATGGCTCCAGTACTTTACAGTCTGAGGGTTCCAACAGTGACATGTATCTCGTGC CTGCTGCCATGTTTCGGGACCCCTTCCGTAAGGACCCTAACAAGCTGGTGTTATGTGAAG TTTTCAAGTACAATCGAAGGCCTGCAGAGACCAATTTGAGGCACACCTGTAAACGGATAA TGGACATGGTGAGCAACCAGCACCCCTGGTTTGGCATGGAGCAGGAGTATACCCTCATGG GGACAGATGGGCACCCCTTTGGTTGGCCTTCCAACGGCTTCCCAGGGCCCCAGGGTCCAT ATTACTGTGGTGTGGGAGCAGACAGAGCCTATGGCAGGGACATCGTGGAGGCCCATTACC GGGCCTGCTTGTATGCTGGAGTCAAGATTGCGGGGACTAATGCCGAGGTCATGCCTGCCC AGTGGGAATTTCAGATTGGACCTTGTGAAGGAATCAGCATGGGAGATCATCTCTGGGTGG CCCGTTTCATCTTGCATCGTGTGTGTGAAGACTTTGGAGTGATAGCAACCTTTGATCCTA AGCCCATTCCTGGGAACTGGAATGGTGCAGGCTGCCATACCAACTTCAGCACCAAGGCCA TGCGGGAGGAGAATGGTCTGAAGTACATCGAGGAGGCCATTGAGAAACTAAGCAAGCGGC ACCAGTACCACATCCGTGCCTATGATCCCAAGGGAGGCCTGGACAATGCCCGACGTCTAA CTGGATTCCATGAAACCTCCAACATCAACGACTTTTCTGGTGGTGTAGCCAATCGTAGCG CCAGCATACGCATTCCCCGGACTGTTGGCCAGGAGAAGAAGGGTTACTTTGAAGATCGTC GCCCCTCTGCCAACTGCGACCCCTTTTTCGGTGACAGAAGCCCTCATCCGCACGTGTCTTC TCAATGAAACCGGCGATGAGCCCTTCCAGTACAAAAATTAAGTGGACTAGACCTCCAGCT GTTGAGCCCCTCCTAGTTCTTCATCCCACTCCAACTCTTCCCCCTCTCCCAGTTGTCCCG ATTGTAACTCAAAGGGTGGAATATCAAGGTCGTTTTTTTTCATTCC SEQ ID NO: 4: GS protein grown in bacteria, used in Example 1 MGSSHHHHHHGGGGSMTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDI VEAHYRACLYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETG DEPFQYKN SEQ ID NO:5 cDNA (optimized cDNA of the bacterium used in Example 1). SEQ ID NO:6 [Lactobacillus acidophilus strain 30SC GS] >tr|F0TG87|F0TG87_LACA3 glutamine synthase OS=Lactobacillus acidophilus (strain 30SC) MSKQYTTEEIRKEVADKDVRFLRLCFTDINGTEKAVEVPTSQLDKVLTNDIRDFDGSSIDGFVRLEESDMVLYPDFSTWSVLPWGDEHGGKIGRLICSVHMTDGKPFAGDPR NNLKRVLGEMKEAGFDTFDIGFEMEFHLFKLDENGNWTTEVPDHASYFDMTSDDEGARCRREIVETLEEIGFEVEAAHHEVGDGQQEIDFRFDDALTTADRCQTFKMVARH IARKHGLFATFMAKPVEGQAGNGMHNNMSLFKNKHNVFYDKDGEFHLSNTALYFLNGILEHARAITAIGNPTVNSYKRLIPGFEAPVYIAWAAKNRSPLVRIPSAGEINTR LEMRSADPTANPYLLLAACLTAGLKGIKEQKMPMKPVEENIFEMTEEERAEHGIKPLPTTLHNAIKAFKEDDLIKSALGEHLTHSFIESKELEWSKYSQSVSDWERQRYMNW SEQ ID NO:7 [Zea mays GS] [corn / Maize GS] >tr|B4G1P1|B4G1P1_MAIZEGlutamine Synthetase MACLTDLVNLSDNTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDCYTPAGEPIPTNKRYNAAKIFSSPEVAAEEPWYGIEQEYTLLQKDTNWPLGWPIGGFPGPQGPYYCGIGAEKSFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISSGDQVWVARYILERITEIAGVVVTFDPKPPIPGDWNGAGAHTNYTESMRKEGGYEVIKAAIEKLKLRHREHIAAYGEGNERRLTGRHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTIIWKP

Claims

1. I) i) a glutamine synthetase protein of SEQ ID NO: 1 or a mutant thereof having glutamine synthetase activity and at least 90% sequence identity with the sequence of SEQ ID NO: 1 and ii) a peptide containing the sequence MGSSHHHHHHGGGGGS at the N-terminus; II) having or containing the sequence of SEQ ID NO: 4; or III) a glutamine synthetase fusion protein encoded by the sequence of SEQ ID NO: 5, and a pharmaceutically acceptable excipient, a pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the mutant of the glutamine synthetase protein of SEQ ID NO: 1 has at least 95% sequence identity with the sequence of SEQ ID NO:

1.

3. The pharmaceutical composition according to claim 2, wherein the mutant of the glutamine synthetase protein of SEQ ID NO: 1 has at least 97%, 98% or 99% sequence identity with the sequence of SEQ ID NO:

1.

4. A pharmaceutical composition comprising a glutamine synthetase fusion protein having the sequence of SEQ ID NO: 4; and a pharmaceutically acceptable excipient.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the glutamine synthetase fusion protein is linked to a moiety, and the moiety is selected from a protein, a peptide, a non-protein polymer or an affinity tag.

6. The pharmaceutical composition according to claim 5, wherein the glutamine synthetase fusion protein is conjugated to polyethylene glycol (PEG).

7. The pharmaceutical composition according to claim 6, wherein PEG is linked to the fusion protein at the N-terminus.

8. The pharmaceutical composition according to claim 7, wherein PEG is 20 kDa aldehyde PEG.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the fusion protein is in a form suitable for systemic parenteral administration.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the glutamine synthetase fusion protein is in a form suitable for parenteral or subcutaneous administration to a subject.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the fusion protein is multimeric or monomeric.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the composition is a parenteral nutritional composition.

13. I) i) a glutamine synthetase protein of SEQ ID NO: 1 or glutamine synthetase activity and a variant thereof having at least 90% sequence identity with the sequence of SEQ ID NO: 1 and ii) a peptide containing the sequence MGSSHHHHHHGGGGGS at the N-terminus; II) having or containing the sequence of SEQ ID NO: 4; or III) a glutamine synthetase fusion protein encoded by the sequence of SEQ ID NO:

5.

14. The glutamine synthetase fusion protein according to claim 13, wherein the variant of the glutamine synthetase protein of SEQ ID NO: 1 has at least 95% sequence identity with the sequence of SEQ ID NO:

1.

15. The glutamine synthetase fusion protein according to claim 14, wherein the variant of the glutamine synthetase protein of SEQ ID NO: 1 has at least 97%, 98% or 99% sequence identity with the sequence of SEQ ID NO:

1.

16. The glutamine synthetase fusion protein according to any one of claims 13 to 15, wherein the glutamine synthetase fusion protein is linked to a moiety, and the moiety is selected from a protein, a peptide, a non-protein polymer or an affinity tag.

17. The glutamine synthetase fusion protein according to claim 16, wherein the glutamine synthetase fusion protein is conjugated to polyethylene glycol (PEG).

18. The glutamine synthetase fusion protein according to claim 17, wherein PEG is linked to the fusion protein at the N-terminus.

19. The glutamine synthetase fusion protein according to claim 18, wherein PEG is 20 kDa aldehyde PEG.

20. Use of a glutamine synthetase fusion protein as defined in any one of claims 13 to 19 for the manufacture of a composition for the treatment or prevention of hyperammonemia by systemic parenteral, parenteral or subcutaneous administration to a subject.

21. The use according to claim 20, wherein the hyperammonemia occurs due to urea cycle disorder (UCD), glutamine synthetase deficiency, non-alcoholic fatty liver disease, hepatic encephalopathy, liver cirrhosis, renal dysfunction, and / or organ failure such as liver failure or renal failure.

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