O-GlcNAcylation enhances CPS1 catalytic efficiency for ammonia and promotes ureagenesis.

Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases.

Intermittent lipid nanoparticle mRNA administration prevents cortical dysmyelination associated with arginase deficiency.

Arginase deficiency is associated with prominent neuromotor features, including spastic diplegia, clonus, and hyperreflexia; intellectual disability and progressive neurological decline are other signs. In a constitutive murine model, we recently described leukodystrophy as a significant component of the central nervous system features of arginase deficiency. In the present studies, we sought to examine if the administration of a lipid nanoparticle carrying human ARG1 mRNA to constitutive knockout mice could prevent abnormalities in myelination associated with arginase deficiency. Imaging of the cingulum, striatum, and cervical segments of the corticospinal tract revealed a drastic reduction of myelinated axons; signs of degenerating axons were also present with thin myelin layers. Lipid nanoparticle/ARG1 mRNA administration resulted in both light and electron microscopic evidence of a dramatic recovery of myelin density compared with age-matched controls; oligodendrocytes were seen to be extending processes to wrap many axons. Abnormally thin myelin layers, when myelination was present, were resolved with intermittent mRNA administration, indicative of not only a greater density of myelinated axons but also an increase in the thickness of the myelin sheath. In conclusion, lipid nanoparticle/ARG1 mRNA administration in arginase deficiency prevents the associated leukodystrophy and restores normal oligodendrocyte function.

Gene therapy for guanidinoacetate methyltransferase deficiency restores cerebral and myocardial creatine while resolving behavioral abnormalities.

Creatine deficiency disorders are inborn errors of creatine metabolism, an energy homeostasis molecule. One of these, guanidinoacetate N-methyltransferase (GAMT) deficiency, has clinical characteristics that include features of autism, self-mutilation, intellectual disability, and seizures, with approximately 40% having a disorder of movement; failure to thrive can also be a component. Along with low creatine levels, guanidinoacetic acid (GAA) toxicity has been implicated in the pathophysiology of the disorder. Present-day therapy with oral creatine to control GAA lacks efficacy; seizures can persist. Dietary management and pharmacological ornithine treatment are challenging. Using an AAV-based gene therapy approach to express human codon-optimized GAMT in hepatocytes, in situ hybridization, and immunostaining, we demonstrated pan-hepatic GAMT expression. Serial collection of blood demonstrated a marked early and sustained reduction of GAA with normalization of plasma creatine; urinary GAA levels also markedly declined. The terminal time point demonstrated marked improvement in cerebral and myocardial creatine levels. In conjunction with the biochemical findings, treated mice gained weight to nearly match their wild-type littermates, while behavioral studies demonstrated resolution of abnormalities; PET-CT imaging demonstrated improvement in brain metabolism. In conclusion, a gene therapy approach can result in long-term normalization of GAA with increased creatine in guanidinoacetate N-methyltransferase deficiency and at the same time resolves the behavioral phenotype in a murine model of the disorder. These findings have important implications for the development of a new therapy for this abnormality of creatine metabolism.

CRISPR-Mediated Genomic Addition to CPS1 Deficient iPSCs is Insufficient to Restore Nitrogen Homeostasis.

CPS1 deficiency is an inborn error of metabolism caused by loss-of-function mutations in the CPS1 gene, catalyzing the initial reaction of the urea cycle. Deficiency typically leads to toxic levels of plasma ammonia, cerebral edema, coma, and death, with the only curative treatment being liver transplantation; due to limited donor availability and the invasiveness and complications of the procedure, however, alternative therapies are needed. Induced pluripotent stem cells offer an alternative cell source to partial or whole liver grafts that theoretically would not require immune suppression regimens and additionally are amenable to genetic modifications. Here, we genetically modified CPS1 deficient patient-derived stem cells to constitutively express human codon optimized CPS1 from the AAVS1 safe harbor site. While edited stem cells efficiently differentiated to hepatocyte-like cells, they failed to metabolize ammonia more efficiently than their unedited counterparts. This unexpected result appears to have arisen in part due to transgene promoter methylation, and thus transcriptional silencing, in undifferentiated cells, impacting their capacity to restore the complete urea cycle function upon differentiation. As pluripotent stem cell strategies are being expanded widely for potential cell therapies, these results highlight the need for strict quality control and functional analysis to ensure the integrity of cell products.

CPS1: Looking at an ancient enzyme in a modern light.

The mammalian urea cycle (UC) is responsible for siphoning catabolic waste nitrogen into urea for excretion. Disruptions of the functions of any of the enzymes or transporters lead to elevated ammonia and neurological injury. Carbamoyl phosphate synthetase 1 (CPS1) is the first and rate-limiting UC enzyme responsible for the direct incorporation of ammonia into UC intermediates. Symptoms in CPS1 deficiency are typically the most severe of all UC disorders, and current clinical management is insufficient to prevent the associated morbidities and high mortality. With recent advances in basic and translational studies of CPS1, appreciation for this enzyme's essential role in the UC has been broadened to include systemic metabolic regulation during homeostasis and disease. Here, we review recent advances in CPS1 biology and contextualize them around the role of CPS1 in health and disease.

Split AAV-Mediated Gene Therapy Restores Ureagenesis in a Murine Model of Carbamoyl Phosphate Synthetase 1 Deficiency.

The urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) catalyzes the initial step of the urea cycle; bi-allelic mutations typically present with hyperammonemia, vomiting, ataxia, lethargy progressing into coma, and death due to brain edema if ineffectively treated. The enzyme deficiency is particularly difficult to treat; early recognition is essential to minimize injury to the brain. Even under optimal conditions, therapeutic interventions are of limited scope and efficacy, with most patients developing long-term neurologic sequelae. One significant encumberment to gene therapeutic development is the size of the CPS1 cDNA, which, at 4.5 kb, nears the packaging capacity of adeno-associated virus (AAV). Herein we developed a split AAV (sAAV)-based approach, packaging the large transgene and its regulatory cassette into two separate vectors, thereby delivering therapeutic CPS1 by a dual vector system with testing in a murine model of the disorder. Cps1-deficient mice treated with sAAVs survive long-term with markedly improved ammonia levels, diminished dysregulation of circulating amino acids, and increased hepatic CPS1 expression and activity. In response to acute ammonia challenging, sAAV-treated female mice rapidly incorporated nitrogen into urea. This study demonstrates the first proof-of-principle that sAAV-mediated therapy is a viable, potentially clinically translatable approach to CPS1 deficiency, a devastating urea cycle disorder.

A constitutive knockout of murine carbamoyl phosphate synthetase 1 results in death with marked hyperglutaminemia and hyperammonemia.

The enzyme carbamoyl phosphate synthetase 1 (CPS1; EC 6.3.4.16) forms carbamoyl phosphate from bicarbonate, ammonia, and adenosine triphosphate (ATP) and is activated allosterically by N-acetylglutamate. The neonatal presentation of bi-allelic mutations of CPS1 results in hyperammonemia with reduced citrulline and is reported as the most challenging nitrogen metabolism disorder to treat. As therapeutic interventions are limited, patients often develop neurological injury or die from hyperammonemia. Survivors remain vulnerable to nitrogen overload, being at risk for repetitive neurological injury. With transgenic technology, our lab developed a constitutive Cps1 mutant mouse and reports its characterization herein. Within 24 hours of birth, all Cps1 -/- mice developed hyperammonemia and expired. No CPS1 protein by Western blot or immunostaining was detected in livers nor was Cps1 mRNA present. CPS1 enzymatic activity was markedly decreased in knockout livers and reduced in Cps1+/- mice. Plasma analysis found markedly reduced citrulline and arginine and markedly increased glutamine and alanine, both intermolecular carriers of nitrogen, along with elevated ammonia, taurine, and lysine. Derangements in multiple other amino acids were also detected. While hepatic amino acids also demonstrated markedly reduced citrulline, arginine, while decreased, was not statistically significant; alanine and lysine were markedly increased while glutamine was trending towards significance. In conclusion we have determined that this constitutive neonatal mouse model of CPS1 deficiency replicates the neonatal human phenotype and demonstrates the key biochemical features of the disorder. These mice will be integral for addressing the challenges of developing new therapeutic approaches for this, at present, poorly treated disorder.

Hepatic glutamine synthetase augmentation enhances ammonia detoxification.

The urea cycle and glutamine synthetase (GS) are the two main pathways for waste nitrogen removal and their deficiency results in hyperammonemia. Here, we investigated the efficacy of liver-specific GS overexpression for therapy of hyperammonemia. To achieve hepatic GS overexpression, we generated a helper-dependent adenoviral (HDAd) vector expressing the murine GS under the control of a liver-specific expression cassette (HDAd-GS). Compared to mice injected with a control vector expressing an unrelated reporter gene (HDAd-alpha-fetoprotein), wild-type mice with increased hepatic GS showed reduced blood ammonia levels and a concomitant increase of blood glutamine after intraperitoneal injections of ammonium chloride, whereas blood urea was unaffected. Moreover, injection of HDAd-GS reduced blood ammonia levels at baseline and protected against acute hyperammonemia following ammonia challenge in a mouse model with conditional hepatic deficiency of carbamoyl phosphate synthetase 1 (Cps1), the initial and rate-limiting step of ureagenesis. In summary, we found that upregulation of hepatic GS reduced hyperammonemia in wild-type and Cps1-deficient mice, thus confirming a key role of GS in ammonia detoxification. These results suggest that hepatic GS augmentation therapy has potential for treatment of both primary and secondary forms of hyperammonemia.

Human hepatocyte transplantation corrects the inherited metabolic liver disorder arginase deficiency in mice.

The transplantation, engraftment, and expansion of primary hepatocytes have the potential to be an effective therapy for metabolic disorders of the liver including those of nitrogen metabolism. To date, such methods for the treatment of urea cycle disorders in murine models has only been minimally explored. Arginase deficiency, an inherited disorder of nitrogen metabolism that presents in the first two years of life, has the potential to be treated by such methods. To explore the potential of this approach, we mated the conditional arginase deficient mouse with a mouse model deficient in fumarylacetoacetate hydrolase (FAH) and with Rag2 and IL2-Rγ mutations to give a selective advantage to transplanted (normal) human hepatocytes. On day -1, a uroplasminogen-expressing adenoviral vector was administered intravenously followed the next day with the transplantation of 1 × 106 human hepatocytes (or vehicle alone) by intrasplenic injection. As the initial number of administered hepatocytes would be too low to prevent hepatotoxicity-induced mortality, NTBC cycling was performed to allow for hepatocyte expansion and repopulation. While all control mice died, all except one human hepatocyte transplanted mice survived. Four months after hepatocyte transplantation, 2 × 1011 genome copies of AAV-TBG-Cre recombinase was administered IV to disrupt endogenous hepatic arginase expression. While all control mice died within the first month, human hepatocyte transplanted mice did well. Ammonia and amino acids, analyzed in both groups before and after disruption of endogenous arginase expression, while well-controlled in the transplanted group, were markedly abnormal in the controls. Ammonium challenging further demonstrated the durability and functionality of the human repopulated liver. In conclusion, these studies demonstrate that human hepatocyte repopulation in the murine liver can result in effective treatment of arginase deficiency.