Stabilized mutant GLUL proteins and uses thereof

Abstract

The present disclosure provides stabilized GLUL mutant proteins and methods of use, e.g., in treating urea cycle disorders, gastrointestinal conditions, and muscle-related disorders.

Description

[0001] PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT STABILIZED MUTANT GLUL PROTEINS AND USES THEREOF

[0002] Cross-Reference to Related Applications

[0003] This application claims the benefit of priority to U.S. Provisional Application No.

[0004] 63 / 737,920, filed on December 23, 2024. The entire contents of the foregoing application are hereby incorporated herein by reference.

[0005] Sequence Listing

[0006] The instant application contains a Sequence Listing which has been filed electronically in extensible Markup Language (XML) format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 24, 2025, is named K199354_1090WO_SL.xml and is 53,221 bytes in size.

[0007] Background

[0008] Ammonia is highly toxic and generated during metabolism in all organs.

[0009] Hyperammonemia is caused by the decreased detoxification and / or increased production of ammonia, in mammals, the urea cycle detoxifies ammonia by enzymatically converting ammonia into urea, which is then removed in the urine. Decreased ammonia detoxification may be caused by urea cycle disorders (UCDs) in which urea cycle enzymes are defective, such as argininosuccinic aciduria, arginase deficiency, carbamoylphosphate synthetase deficiency, citruliinemia, N-acetylglutamate synthetase deficiency, and ornithine transcarbamyiase deficiency. The National Urea Cycle Disorders Foundation estimates that the prevalence of UCDs is 1 in 8,500 births. In addition, several non-UCD disorders, such as hepatic encephalopathy, portosystemic shunting, and organic acid disorders, can also cause hyperammonemia. Hyperammonemia can produce neurological manifestations, e.g.. seizures, ataxia, stroke-like lesions, coma, psychosis, vision loss, acute encephalopathy, cerebral edema, as well as vomiting, respirator alkalosis, hypothermia, or death.

[0010] Ammonia is also a source of nitrogen for amino acids, which are synthesized by various biosynthesis pathways. For example, arginine biosynthesis converts glutamate, which comprises one nitrogen atom, to arginine, which comprises four nitrogen atoms. Intermediate metabolites formed in the arginine biosynthesis pathway, such as citrulline, also incorporate nitrogen. Thus, enhancement of arginine biosynthesis may be used to incorporate excess nitrogen in the body into non-toxic molecules in order to modulate or treat conditions associated with hyperammonemia. Likewise, histidine biosynthesis, methionine biosynthesis, lysine biosynthesis, asparagine biosynthesis, glutamine biosynthesis, and tryptophan biosynthesis arePATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT also capable of incorporating excess nitrogen, and enhancement of those pathways may be used to modulate or treat conditions associated with hyperammonemia.

[0011] Current therapies for hyperammonemia and UCDs aim to reduce ammonia excess, but are widely regarded as suboptimal (Nagamani et al., 2012; Hoffmann et al., 2013; Torres-Vega et al., 2014). Most UCD patients require substantially modified diets consisting of protein restriction. However, a low-protein diet must be carefully monitored; when protein intake is too restrictive, the body breaks down muscle and consequently produces ammonia. In addition, many patients require supplementation with ammonia scavenging drugs, such as sodium phenyl butyrate, sodium benzoate, and glycerol phenylbutyrate, and one or more of these drugs must be administered three to four times per day (Leonard, 2006). Side effects of these drugs include nausea, vomiting, irritability, anorexia, and menstrual disturbance in females (Leonard, 2006). in children, the delivery of food and medication may require a gastrostomy tube surgically implanted in the stomach or a nasogastric tube manually inserted through the nose into the stomach. When these treatment options fail, a liver transplant may be required. Thus, there is significant unmet need for effective, reliable, and / or long-term treatment for disorders associated with hyperammonemia, including urea cycle disorders.

[0012] Summary

[0013] Provided herein are mutant GLUL proteins comprising at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 1 (KI 1R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 2 (KI IE), In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 3 (KI IQ). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 4 (K14R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 5 (K14S). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 6 (K14G). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 7 (K14I). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 8 (K14W). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 9 (K14Y). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 10 (K14E). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 11 (K14Q). In some embodiments, the mutant GLUL protein comprises amino acidPATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT substitutions at both KI 1 and K14. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 12 (KI 1R / K14R). Also provided herein are mutant GLUL proteins that are stable, enzymatically competent, and insensitive to negative feedback by phosphorylation. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 21 (T301 A). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 22 (T301E). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 23 (T301V).

[0014] The disclosure provides methods of treating a urea cycle disorder (UCD) in a subject suffering therefrom comprising exposing the subject to (a) a mutant GLUL protein comprising at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q; or (b) a GLUL Metl8 mutant protein (SEQ ID NO: 13) in an amount sufficient to treat the UCD. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 1 (KI 1R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 2 (KI IE), In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 3 (KI IQ). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 4 (K14R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 5 (K14S). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 6 (K14G). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 7 (K14I). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 8 (K14W). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 9 (K14Y). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 10 (K14E). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 11 (K14Q). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 12 (KI 1R / K14R).

[0015] In another aspect, the disclosure provides methods of reducing ammonia accumulation in a cell comprising contacting the cell with (a) a mutant GLUL protein comprising at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q or (b) a GLUL- Metl8 mutant protein (SEQ ID NO: 13) in an amount sufficient to reduce ammonia accumulation. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 1 (KI 1R). In some embodiments,PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 2 (KI IE), In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 3 (KI IQ). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 4 (K14R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 5 (K14S). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 6 (K14G). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 7 (KI 41). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 8 (K14W). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 9 (K14Y). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 10 (K14E). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 11 (K14Q). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 12 (KI 1R / K14R).

[0016] Also contemplated are methods of reducing ammonia accumulation in a cell, comprising contacting the cell with a nucleotide sequence encoding a mutant GLUL protein comprising at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q, or encoding a GLUL-Metl8 mutant protein (SEQ ID NO: 13), or encoding a GLUL-Met29 mutant protein (SEQ ID NO: 20). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (KI 1R). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 2 (KI IE), In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 3 (KI IQ). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 4 (K14R). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising amino acid sequence set forth in SEQ ID NO: 5 (K14S). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 6 (K14G). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 7 (K14I). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 8 (K14W). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 9 (K14Y). In some embodiments, the nucleotidePATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 10 (K14E). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 11 (K14Q). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 12 (KI 1R / K14R).

[0017] The disclosure also provides methods of treating gastrointestinal conditions characterized by increased intestinal permeability in a subject in need thereof comprising administering to the subject a mutant GLUL protein described herein. In some embodiments, the gastrointestinal condition is selected from irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), Crohn’s disease, celiac disease, or ulcerative colitis. In some embodiments, the mutant GLUL protein comprises at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-12. In some embodiments, the methods comprise administering a GLUL-Metl8 mutant protein (SEQ ID NO: 13), a GLUL-Met29 mutant protein (SEQ ID NO: 20), a GLUL-T301 A mutant protein (SEQ ID NO: 21), a GLUL-T301E mutant protein (SEQ ID NO: 22), or a GLUL-T301 V mutant protein (SEQ ID NO: 23) to the subject. In some embodiments, the mutant GLUL protein exhibits increased stability in the presence of glutamine compared to wild-type GLUL protein, thereby providing sustained glutamine synthesis capacity to restore intestinal barrier function. In some embodiments, the mutant GLUL protein is delivered to intestinal epithelial cells.

[0018] The disclosure further provides methods of treating muscle-related disorders in a subject in need thereof comprising administering to the subject a mutant GLUL protein described herein. In some embodiments, the muscle-related disorder is selected from muscle wasting, cachexia, sarcopenia, impaired muscle regeneration, impaired satellite cell activation, muscle metabolic dysfunction associated with hepatic encephalopathy, muscle metabolic dysfunction associated with cirrhosis, muscle metabolic dysfunction associated with chronic kidney disease, muscle metabolic dysfunction associated with prion diseases, or muscle metabolic dysfunction associated with brown fat thermogenesis. In some embodiments, the mutant GLUL protein comprises at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-12. In some embodiments, the methods comprise administering a GLUL-Metl8 mutant protein (SEQ ID NO: 13), a GLUL-Met29 mutant protein (SEQ ID NO: 20), a GLUL-T301 A mutant protein (SEQ ID NO: 21), a GLUL-T301E mutant protein (SEQ ID NO: 22), or a GLUL-T301 V mutant proteinPATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT (SEQ ID NO: 23) to the subject. In some embodiments, the mutant GLUL protein exhibits increased stability in the presence of glutamine compared to wild-type GLUL protein, thereby providing sustained glutamine synthesis capacity for muscle metabolic homeostasis and protection against catabolic stress. In some embodiments, the mutant GLUL protein is delivered to skeletal muscle tissue.

[0019] Brief Description of the Figures

[0020] Figure l is a graph showing that KI 1R, K14R as well as KI 1R / K14R mutations stabilize GLUL in cells in the presence of 5 mM glutamine in the media.

[0021] FIGs 2A and 2B represent a delta Gibbs free energy change plot for single amino acid substitutions in GLUL modeled in OpenFold against the wild-type GLUL protein modeled in OpenFold. FIG. 2A represents the change in Gibbs free energy change of amino acid substitutions at position 11 of GLUL from wild-type GLUL. FIG. 2B represents the change in Gibbs free energy change of amino acid substitutions at position 14 of GLUL from wild-type GLUL.

[0022] Detailed Description

[0023] The present disclosure is based on the discovery that mutant glutamate-ammonia ligase (GLUL) proteins described herein are stable in the presence of glutamine and maintain GLUL enzymatic activity. In some embodiments, the mutant GLUL protein comprises the substitution of a wild type amino acid of the GLUL protein (Genbank Accession No. NP 002056.2) with an alternative amino acid, i.e., an amino acid that is not a wild type amino acid at a specific position in the GLUL protein that results in a mutant GLUL protein that is stable in the presence of glutamine. The term “mutant GLUL protein” refers to a GLUL protein that includes a mutation at position 11, 14 or both resulting in a mutant GLUL protein that is stable in the presence of glutamine and exhibits GLUL enzymatic activity. Also disclosed is use of a mutant GLUL protein that is Metl8 truncated mutant (i.e., wt GLUL amino acids 1-17 are removed leaving a mutant protein that starts at Metl8 - SEQ ID NO. 13). Also disclosed is use of a mutant GLUL protein that is Met29 truncated mutant (i.e., wt GLUL amino acids 1-28 are removed leaving a mutant protein that starts at Met29 - SEQ ID NO. 20).

[0024] In some embodiments, the mutant GLUL protein comprises at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 1 (KI 1R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 2 (KI IE), In some embodiments, the mutantPATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 3 (KI IQ). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 4 (K14R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 5 (K14S). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 6 (K14G). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 7 (K14I). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 8 (K14W). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 9 (K14Y). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 10 (K14E). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 11 (K14Q). In some embodiments, the mutant GLUL protein comprises amino acid substitutions at both KI 1 and K14. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 12 (KI 1R / K14R). In some embodiments, mutant GLUL protein exhibits increased stability in cells that are in the presence of glutamine as compared to the wild-type protein under similar conditions. Stability of the protein can be assessed in any suitable manner or assay, including as described in the examples below.

[0025] Also disclosed is use of mutant GLUL proteins that contain amino acid substitutions at position 301. The term “GLUL-T301A mutant protein” refers to a GLUL protein that contains a threonine to alanine substitution at position 301 of the wild type GLUL sequence. The GLUL-T301 A mutant protein has been shown to be stable and enzymatically competent yet insensitive to negative feedback by phosphorylation (Huyghe et al., Front. Mol. Neurosci., 12:120, 2019). The term “GLUL-T301E mutant protein” refers to a GLUL protein that contains a threonine to glutamic acid substitution at position 301 of the wild type GLUL sequence. The GLUL-T301E mutant protein has been shown to be stable and enzymatically competent yet insensitive to negative feedback by phosphorylation (Huyghe et al., Front. Mol. Neurosci., 12:120, 2019). The term “GLUL-T301 V mutant protein” refers to a GLUL protein that contains a threonine to valine substitution at position 301 of the wild type GLUL sequence. The GLUL-T301V mutant protein has been shown to be stable and enzymatically competent yet insensitive to negative feedback by phosphorylation (Huyghe et al., Front. Mol. Neurosci., 12:120, 2019).

[0026] Methods of Making a Mutant GLUL Protein

[0027] The mutant GLUL proteins described herein can be produced, for example, by geneediting or mRNA technologies known in the art. For example, mRNA or DNA for GLUL can bePATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT edited by ADAR-editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), additional nucleic acid editing enzymes, e.g., cytidine deaminases (e.g., AP0BEC1 family deaminases), and / or homing endonucleases or meganucleases.

[0028] In some embodiments, the GLUL mRNA is edited by ADAR-editing technology. In mammalian cells, there are three types of ADAR proteins, Adarl (two isoforms, pl 10 and pl 50), Adar2 and Adar3 (catalytically inactive). The catalytic substrate of ADAR protein is doublestranded RNA, and ADAR can remove the -NH2 group from an adenosine (A) nucleobase, changing A to inosine (I). (I) is recognized as guanosine (G) and paired with cytidine (C) during subsequent cellular transcription and translation processes. To achieve targeted RNA editing, the ADAR protein or its catalytic domain can be fused with a kN peptide, a SNAP-tag or a Cas protein (dCasl3b), and a guide RNA can be designed to recruit the chimeric ADAR protein to the target site. Alternatively, overexpressing AD ARI or ADAR2 proteins together with an R / G motif-bearing guide RNA has also been reported to enable targeted RNA editing. ADAR-editing technology is described in more detail in PCT Publication Nos. WO 2020 / 154342, WO 2020 / 154344, and WO 2020 / 154343, the disclosures of which are incorporated herein by reference in their entireties.

[0029] In some embodiments, the GLUL gene is edited by CRISPR technology. CRISPR technology is included in the invention as an approach for generating RNA-guided nuclease with customizable specificities for targeted genome editing. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically.

[0030] In some embodiments, the GLUL gene is edited by the transcription activator like effector nucleases (TALENs). The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site.

[0031] In some embodiments, the GLUL gene is edited by a nucleic acid editing enzyme, e.g., a deaminase, e.g., a cytidine deaminase. The term "cytidine deaminase" or "cytidine deaminase protein" as used herein refers to a protein, a polypeptide, or one or more functional domain(s) of a protein or a polypeptide that is capable of catalyzing a hydrolytic deamination reaction thatPATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT converts an cytosine (or an cytosine moiety of a molecule) to an uracil (or a uracil moiety of a molecule). In some embodiments, the cytosine-containing molecule is a cytidine (C), and the uracil-containing molecule is an uridine (U). The cytosine-containing molecule can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0032] In some embodiments, the cytidine deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase, an activation-induced deaminase (AID), or a cytidine deaminase 1 (CDA1). In some embodiments, the APOBEC family deaminase is selected from the group consisting of APOBEC 1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, or any functional variants or fusion proteins thereof.

[0033] In some embodiments, the cytidine deaminase protein recognizes and converts one or more target cytosine residue(s) in a target RNA or DNA molecule. The changes may be in 5' or 3' untranslated regions of a target RNA, in splice sites, in exons (changing amino acids in protein translated from the target RNA, changing codon usage or splicing behavior by changing exonic splicing silencers or enhancers, and / or introducing or removing start or stop codons), in introns (changing splicing by altering intronic splicing silencers or intronic splicing enhancers, branch points) and in general in any region affecting RNA stability, structure or functioning. The target RNA sequence may comprise a mutation that one may wish to correct or alter, such as a transition or a transversion.

[0034] In certain embodiments, the cytidine deaminase can be introduced into a cell for expression via a viral vector or a non-viral delivery system as described herein or any known viral vectors or non-viral delivery systems in the art.

[0035] Nucleic Acids

[0036] In another aspect, the disclosure provides a nucleic acid molecule encoding a mutant GLUL protein as disclosed herein. The term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be singlestranded or double-stranded.

[0037] In some embodiments, the disclosure provides a nucleic acid molecule (e.g., nucleotide sequence) (e.g., DNA or mRNA) encoding a mutant GLUL protein comprising a substitution at position 11, a substitution at position 14, or substitutions at both positions 11 and 14. In some embodiments, the nucleotide sequence encodes a mutant GLUL protein comprising at least onePATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q. In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (KI 1R). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 2 (KI IE), In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 3 (KI IQ). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 4 (K14R). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising amino acid sequence set forth in SEQ ID NO: 5 (K14S). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 6 (K14G). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 7 (K14I). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 8 (K14W). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 9 (K14Y). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 10 (K14E). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 11 (K14Q).

[0038] In some embodiments, the nucleotide sequence encodes a mutant GLUL protein comprising two amino acid substitutions. In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 12 (KI 1R / K14R).

[0039] A nucleic acid molecule used in the methods of the present disclosure can be isolated using standard molecular biology techniques. Using all or portion of a nucleic acid sequence of interest as a hybridization probe, nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning. A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0040] A nucleic acid molecule can also be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of a nucleic acid molecule of interest. A nucleic acid molecule of the disclosure can be amplified using cDNA,PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT mRNA or, alternatively, genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. Furthermore, oligonucleotides corresponding to nucleotide sequences of interest can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer. In some embodiments, the nucleic acids of the disclosure are prepared by standard recombinant DNA techniques. A nucleic acid of the disclosure can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which has been automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos.

[0041] 4,401,796 and 4,373,071, incorporated by reference herein).

[0042] In one embodiment, the nucleic acid molecule can be present in an inducible construct. In another embodiment, the nucleic acid molecules can be present in a construct which leads to constitutive expression.

[0043] In one embodiment, the nucleic acid molecules of the disclosure may be delivered to a mammalian cell, or to subjects, in a vector, e.g., a recombinant expression vector. In another embodiment, the nucleic acid molecules of the disclosure may be delivered to cells or to subjects, in the absence of a vector.

[0044] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0045] The recombinant expression vectors of the disclosure comprise a nucleic acid of the disclosure in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acidPATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells, those which are constitutively active, those which are inducible, and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the disclosure can be introduced into host cells to thereby produce proteins or portions thereof, including fusion proteins or portions thereof, encoded by nucleic acids as described herein.

[0046] In one embodiment, a nucleic acid molecule encoding a mutant GLUL protein is expressed in mammalian cells using a mammalian expression vector. When used in mammalian cells, the expression vector’s control functions are often provided by viral regulatory elements.

[0047] In some embodiments, the nucleic acid molecule encoding a mutant GLUL protein is contained within a viral vector and may be delivered to cells or to subjects. Exemplary viral vectors include, but are not limited to, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex viral vector, a parvoviral vector, a papillomavirus vector, a vaccinia viral vector, or a hybrid or chimeric vector thereof.

[0048] The vector will include one or more promoters or enhancers, the selection of which will be known to those skilled in the art. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40 (SV40). Suitable promoters include, but are not limited to, the retroviral long terminal repeat (LTR), the SV40 promoter, the human cytomegalovirus (CMV) promoter, and other viral and eukaryotic cellular promoters known to the skilled artisan. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT In another embodiment, the viral vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. In one embodiment, a tissue-specific promoter for use in the vectors and methods of the disclosure is a liver cell-specific promoter.

[0049] Any mammalian cell or cell type susceptible to cell culture, and to expression of polypeptides, may be utilized in accordance with the present disclosure, such as, for example, human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO), monkey kidney (COS), HT1080, CIO, HeLa, baby hamster kidney (BHK), 3T3, C127, CV-1, HaK, NS / 0, and L-929 cells. Non-limiting examples of mammalian cells that may be used in accordance with the present invention include, but are not limited to, BALB / c mouse myeloma line (NSO / 1, ECACC No: 85110503); human retinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidney CV1 line transformed by 5V40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells + / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (M[VIT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells; F54 cells; and a human hepatoma line (Hep G2).

[0050] In another aspect, the present disclosure provides an isolated cell comprising a nucleic acid molecule encoding a mutant GLUL protein comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-12 or (d) a GLUL-Metl8 mutant protein (SEQ ID NO: 13). The term “GLUL-Metl8 mutant protein” refers to a GLUL protein that is truncated at position 18 of the wild type GLUL sequence. The GLUL-Metl8 mutant protein has been shown to be stable and enzymatically competent yet insensitive to negative feedback by glutamine (Jones at al., American J. Hum. Genet., Ill :729-741, 2024). The term “GLUL-Met29 mutant protein” refers to a GLUL protein that is truncated at position 29 of the wild type GLUL sequence. The GLUL-Met29 mutant protein has been shown to be stable and enzymatically competent yet insensitive to negative feedback by glutamine (Jones at al., American J. Hum. Genet., Ill :729-741, 2024).PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT In another aspect, the present disclosure provides a cell modified to express a mutant GLUL protein described herein. In some embodiments, the mutant GLUL protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-12.

[0051] Treatment methods

[0052] The disclosure also provides methods of treating hyperammonemia or a urea cycle disorder (UCD) in a subject in need thereof comprising administering to the subject a mutant GLUL protein described herein. In some embodiments, the mutant GLUL protein comprises at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 1 (KI 1R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 2 (KI IE), In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 3 (KI IQ). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 4 (K14R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 5 (K14S). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 6 (K14G). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 7 (K14I). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 8 (K14W). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 9 (K14Y). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 10 (K14E). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 11 (K14Q). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 12 (KI 1R / K14R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 21 (T301A). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 22 (T301E). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 23 (T301 V). In some embodiments, the method comprises exposing the subject to the mutant GLUL protein (e.g., by delivering to or expressing the mutant GLUL protein in the liver of the subject).

[0053] In some embodiments, the methods described herein comprise administering a GLUL-Metl8 mutant protein to the subject. In some embodiments, the GLUL-Metl8 mutant protein comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, thePATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT methods described herein comprise administering a GLUL-Met29 mutant protein to the subject. In some embodiments, the GLUL-Met29 mutant protein comprises the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the methods described herein comprise administering a GLUL-T301 A mutant protein to the subject. In some embodiments, the GLUL-T301A mutant protein comprises the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the methods described herein comprise administering a GLUL-T301E mutant protein to the subject. In some embodiments, the GLUL-T301E mutant protein comprises the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the methods described herein comprise administering a GLUL-T301 V mutant protein to the subject. In some embodiments, the GLUL-T301 V mutant protein comprises the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the method comprises exposing the subject to the GLUL-Metl8 protein, GLUL-Met29 protein, GLUL-T301A mutant protein, GLUL-T301E mutant protein, or GLUL-T301 V mutant protein (e.g., by delivering to or expressing the GLUL-Metl8 protein, GLUL-Met29 protein, GLUL-T301A mutant protein, GLUL-T301E mutant protein, or GLUL-T301 V mutant protein in the liver of the subject). In some embodiments, the GLUL-Metl8 mutant protein, GLUL-Met29 mutant protein, GLUL-T301A mutant protein, GLUL-T301E mutant protein, or GLUL-T301 V mutant protein is not delivered to or expressed in the CNS of the subject. In some embodiments, the subject does not have encephalopathy due to a start-loss variant in the GLUL gene.

[0054] The terms "hyperammonemia," "hyperammonemia," or "excess ammonia" are used to refer to increased concentrations of ammonia in the body. Hyperammonemia is caused by decreased detoxification and / or increased production of ammonia. Decreased detoxification may result from urea cycle disorders (UCDs), such as argininosuccinic aciduria, arginase deficiency, carbamoylphosphate synthetase deficiency, citrullinemia, N-acetylglutamate synthetase deficiency, and ornithine transcarbamylase deficiency; or from bypass of the liver, e.g., open ductus hepaticus; and / or deficiencies in glutamine synthetase (Hoffman et al., 2013; Haberle et al., 2013). Increased production of ammonia may result from infections, drugs, neurogenic bladder, and intestinal bacterial overgrowth (Haberle et al., 2013). Other disorders and conditions associated with hyperammonemia include, but are not limited to, liver disorders such as hepatic encephalopathy, acute liver failure, or chronic liver failure; organic acid disorders; isovaleric aciduria; 3-methyicrotonyigiycinuria; methylmalonic acidemia; propionic aciduria; fatty acid oxidation defects; carnitine cycle defects; carnitine deficiency; P-oxidation deficiency; lyssnuric protein intolerance; pyrroline-5-carboxylate synthetase deficiency; pyruvate carboxylase deficiency; ornithine aminotransferase deficiency; carbonic anhydrase deficiency;PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT hyperinsulinism-hyperammonemia syndrome; mitochondrial disorders; valproate therapy; asparaginase therapy; total parenteral nutrition; cystoscopy with glycine-containing solutions; post-lung / bone marrow transplantation; portosystemic shunting; urinary tract infections; ureter dilation; multiple myeloma; and chemotherapy (Hoffman et al., 2013; Haberle et al., 2013; Pham et al., 2.013; Lazier et al., 2014). In healthy subjects, plasma ammonia concentrations are typically less than about 50 pmol / L (Leonard, 2006). in some embodiments, a diagnostic signal of hyperammonemia is a plasma ammonia concentration of at least about 50 prmol / L, at least about 80 pmol / L, at least about 150 prmol / L, at least about 180 pmolI / L, or at least about 200 pmolI / L (Leonard, 2006; Hoffman et al., 2013; Haberle et al., 2013).

[0055] The disclosure also provides methods of treating a urea cycle disorder (UCD) in a subject in need thereof, comprising administering to the subject a mutant GLUL protein described herein or a GLUL-Metl8 mutant protein (SEQ ID NO: 13) or a GLUL-Met29 mutant protein (SEQ ID NO: 20) or a GLUL-T301 A mutant protein (SEQ ID NO: 21) or a GLUL-T301E mutant protein (SEQ ID NO: 22) or a GLUL-T301 V mutant protein (SEQ ID NO: 23). In some embodiments, the UCD is a result of a mutation or deficiency in one or more of the following genes: ARG1, CPS1, NAGS, ASS1, SLC25A13, OTC, ASL, and SLC25A15. Exemplary urea cycle disorders include, but are not limited to, argininosuccinic aciduria, arginase deficiency, carbamoylphosphate synthetase deficiency, citrullinemia, N-acetylglutamate synthetase deficiency, and ornithine transcarbamylase deficiency; or open ductus hepaticus; and / or deficiencies in glutamine synthetase. In some embodiments, the mutant GLUL protein for use in the methods described herein comprises at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E andK14Q. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 1 (KI 1R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 2 (KI IE), In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 3 (KI IQ). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 4 (K14R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 5 (K14S). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 6 (K14G). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 7 (K14I). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 8 (K14W). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 9 (K14Y). In some embodiments, the mutant GLUL proteinPATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT comprises the amino acid sequence set forth in SEQ ID NO: 10 (K14E). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 11 (K14Q). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 12 (KI 1R / K14R).

[0056] The disclosure also provides methods of treating irritable bowel syndrome (IBS) and related gastrointestinal conditions characterized by increased intestinal permeability in a subject in need thereof comprising administering to the subject a mutant GLUL protein described herein. IBS patients with increased intestinal permeability have been shown to exhibit decreased GLUL expression and increased miR-29a levels, resulting in impaired glutamine synthesis and compromised intestinal barrier function (Zhou et al., Gut. 2010 June; 59(6): 775-784). In some embodiments, the mutant GLUL protein comprises at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-12. In some embodiments, the methods comprise administering a GLUL-Metl8 mutant protein (SEQ ID NO: 13), a GLUL-Met29 mutant protein (SEQ ID NO: 20), a GLUL-T301 A mutant protein (SEQ ID NO: 21), a GLUL-T301E mutant protein (SEQ ID NO: 22), or a GLUL-T301 V mutant protein (SEQ ID NO: 23) to the subject. Unlike wild-type GLUL protein which is degraded in the presence of glutamine, the mutant GLUL proteins described herein exhibit increased stability in the presence of glutamine, thereby providing sustained glutamine synthesis capacity. Glutamine supplementation has been demonstrated to restore intestinal permeability in IBS patients, and the stabilized mutant GLUL proteins provide a therapeutic approach for maintaining continuous glutamine production to restore and maintain intestinal barrier function. In some embodiments, the method comprises delivering the mutant GLUL protein to intestinal epithelial cells or administering systemically to provide glutamine for intestinal barrier restoration. The related gastrointestinal conditions include but are not limited to inflammatory bowel disease, Crohn’s disease, ulcerative colitis, celiac disease, and other conditions associated with compromised intestinal barrier function and glutamine deficiency.

[0057] The disclosure further provides methods of treating muscle-related disorders by administering the stabilized mutant GLUL proteins described herein to skeletal muscle tissue as a targeted therapeutic approach, wherein the mutant GLUL proteins comprising amino acid substitutions selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E, K14Q, and KI 1R / K14R, or the GLUL-Metl8 mutant protein (SEQ ID NO: 13), GLUL-Met29 mutant protein (SEQ ID NO: 20), T301 A mutant protein (SEQ ID NO: 21), T301E mutant protein (SEQ ID NO: 22), or T301 V mutant protein (SEQ ID NO: 23) provide sustainedPATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT glutamine synthetase activity that is resistant to glutamine-mediated degradation. In muscle regeneration and satellite cell activation, the stabilized mutant GLUL proteins enhance macrophage-derived glutamine availability for mTOR pathway activation in satellite cells as demonstrated by Shang et al. (Nature 2020), wherein the glutamine-insensitive properties of the mutant proteins maintain continuous glutamine synthesis capacity that would otherwise be diminished by negative feedback inhibition in the presence of elevated glutamine concentrations during the regenerative process. The mutant GLUL proteins are particularly effective in preventing muscle wasting in catabolic states, including cachexia where GLUL upregulation correlates with preservation of muscle mass as shown by Campelj et al. (J Cachexia Sarcopenia Muscle 2025), and sarcopenia as demonstrated by Wu et al. (Nutrients 2024), wherein the sustained enzymatic activity of the mutant proteins addresses the metabolic demands of catabolic stress that would otherwise overwhelm wild-type GLUL through glutamine-mediated protein degradation. For ammonia detoxification in liver disease, the stabilized mutant GLUL proteins maintain ammonia clearance function in skeletal muscle during hepatic encephalopathy and cirrhosis as described by Kircheis & Luth (Drugs 2019), with the glutamine-stable variants providing continuous ammonia sequestration capacity as demonstrated by Holeck & Vodenicarovova (Int J Exp Pathol 2019) in cirrhotic conditions and Aamann et al. (Liver Int 2019) in bile duct-ligated models, wherein the mutant proteins resist the glutamine-mediated degradation that limits wild-type GLUL effectiveness during periods of elevated ammonia and glutamine concentrations. The mutant GLUL proteins provide enhanced metabolic protection through sustained glutamine synthetase activity during brown fat thermogenesis as described by Park et al. (Nature Metabolism 2023), preventing ammonia accumulation while supporting fuel oxidation, and maintain protein content regulation as shown by de Vasconcelos et al. (Cell Physiol Biochem 2019), wherein the glutamine-insensitive properties ensure continuous intracellular glutamine availability for protein homeostasis. The stabilized mutant proteins support the glutamine-glutathione axis for cytoprotection as demonstrated by Leite et al. (Appl Physiol Nutr Metab 2016), providing sustained glutamine synthesis capacity that maintains glutathione production and reduces oxidative stress through continuous enzymatic activity that is not subject to glutamine-mediated feedback inhibition. In disease-specific applications, the mutant GLUL proteins address the altered glutamate / glutamine metabolism observed in prion diseases as described by Caredio et al. (PLoS Pathog 2024) and the metabolic disturbances in chronic kidney disease as shown by Murtas et al. (J Ren Nutr 2020), wherein the stability of the mutant proteins under pathological glutamine concentrations provides therapeutic advantage over wild-type GLUL. The specific amino acid substitutions at positions KI 1 and K14, the N-PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT terminal truncations in GLUL-Metl8 and GLUL-Met29, and the T301 substitutions in the T301 A, T301E, and T301 V variants confer superior therapeutic properties for muscle-targeted therapy by maintaining enzymatic competence in the presence of elevated glutamine concentrations that would otherwise degrade or inhibit wild-type GLUL protein, thereby providing sustained glutamine synthesis capacity essential for muscle metabolic homeostasis, regeneration, and protection against catabolic stress.

[0058] In some embodiments, the methods described herein comprise administering a GLUL-Metl8 mutant protein to the subject. In some embodiments, the GLUL-Metl8 mutant protein comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the methods described herein comprise administering a GLUL-Met29 mutant protein to the subject. In some embodiments, the GLUL-Met29 mutant protein comprises the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the methods described herein comprise administering a GLUL-T301 A mutant protein to the subject. In some embodiments, the GLUL-T301A mutant protein comprises the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the methods described herein comprise administering a GLUL-T301E mutant protein to the subject. In some embodiments, the GLUL-T301E mutant protein comprises the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the methods described herein comprise administering a GLUL-T301 V mutant protein to the subject. In some embodiments, the GLUL-T301 V mutant protein comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0059] The disclosure also provides methods of reducing ammonia accumulation in a cell, comprising contacting the cell with a nucleotide sequence encoding an amino acid sequence set forth in any one of SEQ ID NOs: 1-12. In some embodiments, the nucleotide sequence encodes a mutant GLUL protein comprising at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E andK14Q. In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (KI 1R). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 2 (KI IE), In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 3 (KI IQ). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 4 (K14R). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising amino acid sequence set forth in SEQ ID NO: 5 (K14S). In some embodiments, the nucleotide sequence encodes the mutant GLULPATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT protein comprising the amino acid sequence set forth in SEQ ID NO: 6 (K14G). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 7 (K14I). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 8 (K14W). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 9 (K14Y). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 10 (K14E). In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 11 (K14Q).

[0060] In some embodiments, the nucleotide sequence encodes a mutant GLUL protein comprising two amino acid substitutions. In some embodiments, the nucleotide sequence encodes the mutant GLUL protein comprising the amino acid sequence set forth in SEQ ID NO: 12 (KI 1R / K14R).

[0061] The terms "treat," "treated," or "treating" mean both therapeutic treatment and prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0062] In some embodiments, the methods described herein reduce ammonia concentrations in a subject. In some embodiments, the methods described herein reduce the ammonia concentration in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, reduction is measured by comparing the ammonia concentration in a subject before and after administration of the mutant GLUL protein (or GLUL-Metl8 mutant protein). In some embodiments, the methods described herein allow one or more symptoms of the conditionPATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT or disorder to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0063] Before, during, and after the administration of a mutant GLUL protein (or GLUL-Metl8 mutant protein) described herein, ammonia concentrations in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and / or a sample collected from the contents of one or more of the following: the stomach, duodenum jejunum, ileum, cecum, colorectum, and anal canal. In some embodiments, the methods described herein reduce ammonia concentrations in a subject to undetectable levels, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject's ammonia concentrations prior to treatment.

[0064] Pharmaceutical Composition and Routes of Administration

[0065] The mutant GLUL proteins (or nucleic acid sequences encoding the mutant GLUL proteins) described herein can be administered to a subject in need thereof by any route. Thus, as appropriate, administration can be oral or parenteral, including intravenous, intravitreal, and intraperitoneal routes of administration.

[0066] Suitable oral dosage forms for the compositions described herein include tablets, capsules, solutions, suspensions, syrups, lozenges, and dry powders. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.

[0067] In some embodiments, the composition further comprises an acceptable carrier. The term "carrier" includes, but is not limited to, lipids, phospholipids, salts, emulsifiers, excipients, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof. Polymers used in the dosage form include hydrophobic or hydrophilic polymers and pH dependent or independent polymers. Exemplary hydrophobic and hydrophilic polymers include, but are not limited to, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxy methylcellulose, polyethylene glycol, ethylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate, and ion exchange resins. “Carrier” also includes all components of the coating composition, which may include plasticizers, pigments, colorants, stabilizing agents, and glidants.PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT The compositions described herein can be prepared using one or more excipients, including diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.

[0068] Embodiments:

[0069] Provided herein are mutant GLUL proteins comprising at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 1 (KI 1R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 2 (KI IE). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 3 (KI IQ). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 4 (K14R). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 5 (K14S). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 6 (K14G). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 7 (K14I). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 8 (K14W). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 9 (K14Y). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 10 (K14E). In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 11 (K14Q). In some embodiments, the mutant GLUL protein comprises amino acid substitutions at both KI 1 and K14. In some embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 12 (KI 1R / K14R).

[0070] In some embodiments, the mutant GLUL protein exhibits increased stability in cells that are in the presence of glutamine as compared to the wild-type protein under similar conditions. In further embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 21 (T301 A). In other embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 22 (T301E). In still other embodiments, the mutant GLUL protein comprises the amino acid sequence set forth in SEQ ID NO: 23 (T301V).

[0071] Also provided herein are nucleic acid molecules encoding the mutant GLUL proteins described above. In some embodiments, the disclosure provides a vector comprising such nucleic acid molecules. In some embodiments, the vector is a viral vector or a non-viral vector. In further embodiments, the viral vector is selected from the group consisting of an adenoviral vector, anPATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex viral vector, a parvoviral vector, a papillomavirus vector, a vaccinia viral vector, or a hybrid or chimeric vector thereof.

[0072] The disclosure also provides methods of treating hyperammonemia or a urea cycle disorder (UCD) in a subject suffering therefrom comprising exposing the subject to (a) a mutant GLUL protein comprising at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q, (b) a GLUL-Metl8 mutant protein (SEQ ID NO: 13), (c) a GLUL-Met29 mutant protein (SEQ ID NO: 20), (d) a GLUL-T301 A mutant protein (SEQ ID NO: 21), (e) a GLUL-T301E mutant protein (SEQ ID NO: 22), or (f) a GLUL-T301 V mutant protein (SEQ ID NO: 23) in an amount sufficient to treat the hyperammonemia or UCD. In some embodiments, the method comprises exposing the subject to the mutant GLUL protein comprising at least one amino acid substitution at KI 1 or K14. In other embodiments, the method comprises exposing the subject to a GLUL-Metl8 mutant protein, a GLUL-Met29 mutant protein, a GLUL-T301 A mutant protein, a GLUL-T301E mutant protein, or a GLUL-T301 V mutant protein.

[0073] In some embodiments, exposing the subject to a mutant GLUL protein, GLUL-Metl8 mutant protein, GLUL-Met29 mutant protein, GLUL-T301 A mutant protein, GLUL-T301E mutant protein, or GLUL-T301 V mutant protein is done by delivering to or expressing in the liver of the subject. In further embodiments, the mutant GLUL protein, GLUL-Metl8 mutant protein, GLUL-Met29 mutant protein, GLUL-T301 A mutant protein, GLUL-T301E mutant protein, or GLUL-T301 V mutant protein is delivered to, or expressed in liver hepatocytes. In some embodiments, the mutant GLUL protein, GLUL-Metl8 mutant protein, GLUL-Met29 mutant protein, GLUL-T301 A mutant protein, GLUL-T301E mutant protein, or GLUL-T301 V mutant protein is not delivered to or expressed in the CNS of the subject.

[0074] In some embodiments, the UCD is a result of a mutation or deficiency in one or more of ARG1, CPS1, NAGS, ASS1, SLC25A13, OTC, ASL, and SLC25A15. In further embodiments, the UCD is argininosuccinic aciduria, arginase deficiency, carbamoylphosphate synthetase deficiency, citrullinemia, N-acetylglutamate synthetase deficiency, and ornithine transcarbamylase deficiency; or open ductus hepaticus; and / or deficiencies in glutamine synthetase. In some embodiments, the subject does not have encephalopathy due to a start-loss variant in GLUL gene.

[0075] The disclosure also provides an isolated cell modified to express the mutant GLUL protein described herein. Additionally, the disclosure provides methods of reducing ammoniaPATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT accumulation in a cell comprising contacting the cell with (a) a mutant GLUL protein comprising at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q, (b) a GLUL Metl8 mutant protein (SEQ ID NO: 13), (c) a GLUL-Met29 mutant protein (SEQ ID NO: 20), (d) a GLUL-T301 A mutant protein (SEQ ID NO: 21), (e) a GLUL-T301E mutant protein (SEQ ID NO: 22), or (f) a GLUL-T301 V mutant protein (SEQ ID NO: 23).

[0076] The disclosure further provides methods of treating a gastrointestinal condition characterized by increased intestinal permeability in a subject suffering therefrom comprising administering to the subject (a) a mutant GLUL protein comprising at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q, (b) a GLUL-Metl8 mutant protein (SEQ ID NO: 13), (c) a GLUL-Met29 mutant protein (SEQ ID NO: 20), (d) a GLUL-T301 A mutant protein (SEQ ID NO: 21), (e) a GLUL-T301E mutant protein (SEQ ID NO: 22), or (f) a GLUL-T301 V mutant protein (SEQ ID NO: 23) in an amount sufficient to restore intestinal barrier function. In some embodiments, the gastrointestinal condition is selected from irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), Crohn’s disease, celiac disease, or ulcerative colitis.

[0077] In some embodiments, the mutant GLUL protein exhibits increased stability in the presence of glutamine compared to wild-type GLUL protein, thereby providing sustained glutamine synthesis capacity for intestinal barrier restoration. In further embodiments, the mutant GLUL protein is delivered to intestinal epithelial cells.

[0078] The disclosure also provides methods of treating muscle-related disorders in a subject suffering therefrom comprising administering to the subject (a) a mutant GLUL protein comprising at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q, (b) a GLUL-Metl8 mutant protein (SEQ ID NO: 13), (c) a GLUL-Met29 mutant protein (SEQ ID NO: 20), (d) a GLUL-T301 A mutant protein (SEQ ID NO: 21), (e) a GLUL-T301E mutant protein (SEQ ID NO: 22), or (f) a GLUL-T301V mutant protein (SEQ ID NO: 23) in an amount sufficient to treat the muscle-related disorder. In some embodiments, the muscle-related disorder is selected from muscle wasting, cachexia, sarcopenia, impaired muscle regeneration, impaired satellite cell activation, muscle metabolic dysfunction associated with hepatic encephalopathy, muscle metabolic dysfunction associated with cirrhosis, muscle metabolic dysfunction associated with chronic kidney disease, muscle metabolic dysfunction associated with prion diseases, or muscle metabolic dysfunction associated with brown fat thermogenesis.PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT In some embodiments, the mutant GLUL protein exhibits increased stability in the presence of glutamine compared to wild-type GLUL protein, thereby providing sustained glutamine synthesis capacity for muscle metabolic homeostasis and protection against catabolic stress. In further embodiments, the mutant GLUL protein is delivered to skeletal muscle tissue.

[0079] EXAMPLES

[0080] Example 1

[0081] Hep3B cells were transfected with the individual plasmids encoding GLUL-K11R (SEQ ID NO: 14) or GLUL-K14R (SEQ ID NO: 15). Hep3B cells were plated at 600,000 per well in a 6 well plate. The cells were first plated in media without glutamine and then 48hr later, the media was supplemented with 5mM glutamine for four hours to induce degradation. As shown in FIG 1, both the KI 1R and K14R as well as the KI 1R / K14R mutations inhibited GLUL degradation in the presence of glutamine.

[0082] Example 2

[0083] Building on the observation that GLUL proteins individually comprising KII R and K14R exhibited increased stability in the presence of glutamine in the media, the likelihood that other amino acid substitutions at the KI 1 or K14 positions would also increase stability of GLUL proteins was assessed. Each of the wild-type protein and KI 1R and K14R substitutions were modelled in the GLUL protein structure using OpenFold (Ahldritz et al., Nat. Methods, 21:1514-1524, 2024). Once modelled, the Gibbs free energy change from the wild-type protein were calculated for the KI 1R and K14R substitution variants (AGmutant - AGWT = AAG). Using this benchmark, other possible amino acid substitutions were modeled in a similar fashion in OpenFold. Once these were modeled, the Gibbs free energy change from the wild-type protein were also calculated. The Gibbs free energy change from wild-type was then plotted. Using the premise that the KI 1R and K14R were stabilizing mutations, any other substitutions that exhibit a Gibbs free energy smaller than these at each position should also stabilize the GLUL protein. Results show that KI IE and KI IQ (FIG 2A) and K14S, K14G, K14I, K14W, K14Y, K14E and K14Q (FIG. 2B) exhibited a Gibbs free energy smaller the KI 1R and K14R substitutions and is also likely stabilize GLUL in the presence of glutamine.

[0084] References:

[0085] Haberle et al. Suggested guidelines for the diagnosis and management of urea cycle disorders. Orphanet J Rare Dis. 2012 May 29;7:32. Review, PM ID: 22642880;PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT Haberle J. Clinical and biochemical aspects of primary and secondary hyperammonemic disorders. Arch Biochem Biophys, 2013 Aug 15;536(2): 101-8. Review. PMID: 23628343;

[0086] Hoffmann et al. Defects in amino acid catabolism and the urea cycle. Handb Clin Neurol.

[0087] 2013;113:1755-73. Review. PMID: 23622399;

[0088] Leonard (2006). Disorders of the urea cycle and related enzymes. Inborn Metabolic Diseases, 4tn ed (pp. 263-272). Springer edizin Verlag Heidelberg;

[0089] Nagamani et al. Optimizing therapy for argininosuccinic aciduria. Mol Genet Metab.

[0090] 2012 Sep; 107(1-2): 10-4. Review. PMID: 22841516;

[0091] Torres-Vega et al. Delivery of giutamine synthetase gene by baculovirus vectors: a proof of concept for the treatment of acute hyperammonemia. Gene Ther. 2014 Oct 23;22(l):58-64, PMID: 25338921;

[0092] Walker, Severe hyperammonaemia in adults not explained by liver disease. Ann Clin Biochem. 2012 May;49(Pt 3):214-28. Review. PMID: 22349554

[0093] Informal Sequence Listing

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Claims

PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT CLAIMS1. A mutant GLUL protein comprising at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q.

2. The mutant GLUL protein of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 1 (KI 1R).

3. The mutant GLUL protein of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 2 (KI IE).

4. The mutant GLUL protein of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 3 (KI IQ).

5. The mutant GLUL protein of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 4 (K14R).

6. The mutant GLUL protein of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 5 (K14S).

7. The mutant GLUL protein of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 6 (K14G).

8. The mutant GLUL protein of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 7 (KI 41).

9. The mutant GLUL protein of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 8 (K14W).

10. The mutant GLUL protein of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 9 (K14Y).

11. The mutant GLUL protein of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 10 (K14E).

12. The mutant GLUL protein of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 11 (K14Q).

13. The mutant GLUL protein of any one of claims 1-12, comprising amino acid substitutions at both KI 1 and K14.

14. The mutant GLUL protein of claim 13, comprising the amino acid sequence set forth in SEQ ID NO: 12 (KI 1R / K14R).

15. The mutant GLUL protein of any one of claims 1-14, wherein the protein exhibits increased stability in cells that are in the presence of glutamine as compared to the wild-type protein under similar conditions.PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT 16. A nucleic acid molecule encoding an amino acid sequence set forth in in any one of claims 1-15.

17. A vector comprising the nucleic acid molecule of claim 16.

18. The vector of claim 17, wherein the vector is a viral vector, or a non-viral vector.

19. The vector of claim 18, wherein the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex viral vector, a parvoviral vector, a papillomavirus vector, a vaccinia viral vector, or a hybrid or chimeric vector thereof.

20. A method of treating hyperammonemia or a urea cycle disorder (UCD) in a subject suffering therefrom comprising exposing the subject to (a) the mutant GLUL protein of any one of claims 1 to 15, (b) a GLUL-Metl8 mutant protein (SEQ ID NO: 13), (c) a GLUL-Met29 mutant protein (SEQ ID NO: 20), (d) a GLUL-T301 A mutant protein (SEQ ID NO: 21), (e) a GLUL-T301E mutant protein (SEQ ID NO: 22), or (f) a GLUL-T301 V mutant protein (SEQ ID NO: 23) in an amount sufficient to treat the hyperammonemia or UCD.

21. The method of claim 20, comprising exposing the subject to the mutant GLUL protein of any one of claims 1 to 15.

22. The method of claim 20, comprising exposing the subject to a GLUL-Metl8 mutant protein (SEQ ID NO: 13), a GLUL-Met29 mutant protein (SEQ ID NO: 20), a GLUL-T301 A mutant protein (SEQ ID NO: 21), a GLUL-T301E mutant protein (SEQ ID NO: 22), or a GLUL-T301 V mutant protein (SEQ ID NO: 23).

23. The method of claim 21 or 22, wherein exposing the subject to a mutant GLUL protein, GLUL-Metl8 mutant protein, GLUL-Met29 mutant protein, GLUL-T301A mutant protein, GLUL-T301E mutant protein, or GLUL-T301V mutant protein is done by delivering to or expressing in the liver of the subject.

24. The method of claim 23, wherein the mutant GLUL protein, GLUL-Metl8 mutant protein, GLUL-Met29 mutant protein, GLUL-T301 A mutant protein, GLUL-T301E mutant protein, or GLUL-T301 V mutant protein is delivered to, or expressed in liver hepatocytes.

25. The method of any one of claims 20-24, wherein the mutant GLUL protein, GLUL-Metl8 mutant protein, GLUL-Met29 mutant protein, GLUL-T301A mutant protein, GLUL-T301E mutant protein, or GLUL-T301 V mutant protein is not delivered to or expressed in the CNS of the subject.

26. The method of any one of claims 20-25, wherein the UCD is a result of a mutation or deficiency in one or more of ARG1, CPS1, NAGS, ASS1, SLC25A13, OTC, ASL, and SLC25A15.PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT 27. The method of any one of claims 20-26, wherein the UCD is argininosuccinic aciduria, arginase deficiency, carbamoylphosphate synthetase deficiency, citrullinemia, N-acetylglutamate synthetase deficiency, and ornithine transcarbamylase deficiency; or open ductus hepaticus; and / or deficiencies in glutamine synthetase.

28. The method of any one of claims 20-27, wherein the subject does not have encephalopathy due to a start-loss variant in GLUL gene.

29. An isolated cell modified to express the mutant GLUL protein of any one of claims 1-15.

30. A method of reducing ammonia accumulation in a cell comprising contacting the cell with (a) the mutant GLUL protein of any one of claims 1-15 or, (b) a GLUL Metl8 mutant protein (SEQ ID NO: 13), (c) a GLUL-Met29 mutant protein (SEQ ID NO: 20), (d) a GLUL-T301 A mutant protein (SEQ ID NO: 21), (e) a GLUL-T301E mutant protein (SEQ ID NO: 22), or (f) a GLUL-T301 V mutant protein (SEQ ID NO: 23).

31. The method of claim 30, comprising contacting the cell with the mutant GLUL protein is the mutant GLUL protein of any one of claims 1 to 15.

32. The method of claim 30, comprising contacting the cell with a GLUL Metl8 mutant protein (SEQ ID NO: 13), a GLUL-Met29 mutant protein (SEQ ID NO: 20), a GLUL-T301 A mutant protein (SEQ ID NO: 21), a GLUL-T301E mutant protein (SEQ ID NO: 22), or a GLUL-T301 V mutant protein (SEQ ID NO: 23).

33. A method of reducing ammonia accumulation in a cell, comprising contacting the cell with a nucleotide sequence encoding an amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

34. A method of treating a gastrointestinal condition characterized by increased intestinal permeability in a subject suffering thereof comprising administering to the subject (a) a mutant GLUL protein comprising at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E andK14Q, (b) a GLUL-Metl8 mutant protein (SEQ ID NO: 13), (c) a GLUL-Met29 mutant protein (SEQ ID NO: 20), (d) a GLUL-T301 A mutant protein (SEQ ID NO: 21), (e) a GLUL-T301E mutant protein (SEQ ID NO: 22), or (f) a GLUL-T301 V mutant protein (SEQ ID NO: 23) in an amount sufficient to restore intestinal barrier.

35. The method of claim 34, wherein the gastrointestinal condition is selected from function. irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), Crohn’s disease, celiac disease, or ulcerative colitis.PATENT ATTORNEY DOCKET NO.: K199354 1090WO / KB-043-PCT 36. The method of any one of claims 34-35, wherein the mutant GLUL protein exhibits increased stability in the presence of glutamine compared to wild-type GLUL protein, thereby providing sustained glutamine synthesis capacity for intestinal barrier restoration.

37. The method of any one of claims 34-36, wherein the mutant GLUL protein is delivered to intestinal epithelial cells.

38. A method of treating a muscle-related disorder in a subject suffering therefrom comprising administering to the subject (a) a mutant GLUL protein comprising at least one amino acid substitution selected from KI 1R, KI IE, KI IQ, K14R, K14S, K14G, K14I, K14W, K14Y, K14E and K14Q, (b) a GLUL-Metl8 mutant protein (SEQ ID NO: 13), (c) a GLUL-Met29 mutant protein (SEQ ID NO: 20), (d) a GLUL-T301 A mutant protein (SEQ ID NO: 21), (e) a GLUL-T301E mutant protein (SEQ ID NO: 22), or (f) a GLUL-T301 V mutant protein (SEQ ID NO: 23) in an amount sufficient to treat the muscle-related disorder.

39. The method of claim 38 wherein the muscle-related disorder is selected from muscle wasting, cachexia, sarcopenia, impaired muscle regeneration, impaired satellite cell activation, muscle metabolic dysfunction associated with hepatic encephalopathy, muscle metabolic dysfunction associated with cirrhosis, muscle metabolic dysfunction associated with chronic kidney disease, muscle metabolic dysfunction associated with prion diseases, or muscle metabolic dysfunction associated with brown fat thermogenesis.

40. The method of any one of claims 38-39, wherein the mutant GLUL protein exhibits increased stability in the presence of glutamine compared to wild-type GLUL protein, thereby providing sustained glutamine synthesis capacity for muscle metabolic homeostasis and protection against catabolic stress.

41. The method of any one of claims 38-40, wherein the mutant GLUL protein is delivered to skeletal muscle tissue.

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