Argininosuccinate lyase drives activation of mutant TERT promoter in glioblastomas.

Cancer-specific TERT promoter mutations have been linked to the reactivation of epigenetically silenced TERT gene by creating de novo binding motifs for E-Twenty-Six transcription factors, especially GABPA. How these mutations switch on TERT from epigenetically repressed states to expressed states have not been defined. Here, we revealed that EGFR activation induces ERK1/2-dependent phosphorylation of argininosuccinate lyase (ASL) at Ser417 (S417), leading to interactions between ASL and GABPA at the mutant regions of TERT promoters. The ASL-generated fumarate inhibits KDM5C, leading to enhanced trimethylation of histone H3 Lys4 (H3K4me3), which in turn promotes the recruitment of c-Myc to TERT promoters for TERT expression. Expression of ASL S417A, which abrogates its binding with GABPA, results in reduced TERT expression, inhibited telomerase activity, shortened telomere length, and impaired brain tumor growth in mice. This study reveals an unrecognized mechanistic insight into epigenetically activation of mutant TERT promoters where GABPA-interacted ASL plays an instrumental role.

Genetic and functional correction of argininosuccinate lyase deficiency using CRISPR adenine base editors.

Argininosuccinate lyase deficiency (ASLD) is a recessive metabolic disorder caused by variants in ASL. In an essential step in urea synthesis, ASL breaks down argininosuccinate (ASA), a pathognomonic ASLD biomarker. The severe disease forms lead to hyperammonemia, neurological injury, and even early death. The current treatments are unsatisfactory, involving a strict low-protein diet, arginine supplementation, nitrogen scavenging, and in some cases, liver transplantation. An unmet need exists for improved, efficient therapies. Here, we show the potential of a lipid nanoparticle-mediated CRISPR approach using adenine base editors (ABEs) for ASLD treatment. To model ASLD, we first generated human-induced pluripotent stem cells (hiPSCs) from biopsies of individuals homozygous for the Finnish founder variant (c.1153C>T [p.Arg385Cys]) and edited this variant using the ABE. We then differentiated the hiPSCs into hepatocyte-like cells that showed a 1,000-fold decrease in ASA levels compared to those of isogenic non-edited cells. Lastly, we tested three different FDA-approved lipid nanoparticle formulations to deliver the ABE-encoding RNA and the sgRNA targeting the ASL variant. This approach efficiently edited the ASL variant in fibroblasts with no apparent cell toxicity and minimal off-target effects. Further, the treatment resulted in a significant decrease in ASA, to levels of healthy donors, indicating restoration of the urea cycle. Our work describes a highly efficient approach to editing the disease-causing ASL variant and restoring the function of the urea cycle. This method relies on RNA delivered by lipid nanoparticles, which is compatible with clinical applications, improves its safety profile, and allows for scalable production.

Nitric oxide is required for lung alveolarization revealed by deficiency of argininosuccinate lyase.

Inhaled nitric oxide (NO) therapy has been reported to improve lung growth in premature newborns. However, the underlying mechanisms by which NO regulates lung development remain largely unclear. NO is enzymatically produced by three isoforms of nitric oxide synthase (NOS) enzymes. NOS knockout mice are useful tools to investigate NO function in the lung. Each single NOS knockout mouse does not show obvious lung alveolar phenotype, likely due to compensatory mechanisms. While mice lacking all three NOS isoforms display impaired lung alveolarization, implicating NO plays a pivotal role in lung alveolarization. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing L-arginine, the sole precursor for NOS-dependent NO synthesis. ASL is also required for channeling extracellular L-arginine into a NO-synthetic complex. Thus, ASL deficiency (ASLD) is a non-redundant model for cell-autonomous, NOS-dependent NO deficiency. Here, we assessed lung alveolarization in ASL-deficient mice. Hypomorphic deletion of Asl (AslNeo/Neo) results in decreased lung alveolarization, accompanied with reduced level of S-nitrosylation in the lung. Genetic ablation of one copy of Caveolin-1, which is a negative regulator of NO production, restores total S-nitrosylation as well as lung alveolarization in AslNeo/Neo mice. Importantly, NO supplementation could partially rescue lung alveolarization in AslNeo/Neo mice. Furthermore, endothelial-specific knockout mice (VE-Cadherin Cre; Aslflox/flox) exhibit impaired lung alveolarization at 12 weeks old, supporting an essential role of endothelial-derived NO in the enhancement of lung alveolarization. Thus, we propose that ASLD is a model to study NO-mediated lung alveolarization.

Arginine inhibition of the argininosuccinate lyases is conserved among three orders in cyanobacteria.

KEY MESSAGE: This study revealed different catalytic efficiencies of cyanobacterial argininosuccinate lyases in non-nitrogen-fixing and nitrogen-fixing cyanobacteria, demonstrating that L-arginine inhibition of L-argininosuccinate lyase is conserved among enzymes of three cyanobacterial orders. Arginine is a nitrogen-rich amino acid that uses a nitrogen reservoir, and its biosynthesis is strictly controlled by feedback inhibition. Argininosuccinate lyase (EC 4.3.2.1) is the final enzyme in arginine biosynthesis that catalyzes the conversion of argininosuccinate to L-arginine and fumarate. Cyanobacteria synthesize intracellular cyanophycin, which is a nitrogen reservoir composed of aspartate and arginine. Arginine is an important source of nitrogen for cyanobacteria. We expressed and purified argininosuccinate lyases, ArgHs, from Synechocystis sp. PCC 6803, Nostoc sp. PCC 7120, and Arthrospira platensis NIES-39. The catalytic efficiency of the Nostoc sp. PCC 7120 ArgH was 2.8-fold higher than those of Synechocystis sp. PCC 6803 and Arthrospira platensis NIES-39. All three ArgHs were inhibited in the presence of arginine, and their inhibitory effects were lowered at pH 7.0, compared to those at pH 8.0. These results indicate that arginine inhibition of ArgH is widely conserved among the three cyanobacterial orders. The current results demonstrate the conserved regulation of enzymes in the cyanobacterial aspartase/fumarase superfamily.

ASL expression in ALDH1A1+ neurons in the substantia nigra metabolically contributes to neurodegenerative phenotype.

Argininosuccinate lyase (ASL) is essential for the NO-dependent regulation of tyrosine hydroxylase (TH) and thus for catecholamine production. Using a conditional mouse model with loss of ASL in catecholamine neurons, we demonstrate that ASL is expressed in dopaminergic neurons in the substantia nigra pars compacta, including the ALDH1A1 + subpopulation that is pivotal for the pathogenesis of Parkinson disease (PD). Neuronal loss of ASL results in catecholamine deficiency, in accumulation and formation of tyrosine aggregates, in elevation of α-synuclein, and phenotypically in motor and cognitive deficits. NO supplementation rescues the formation of aggregates as well as the motor deficiencies. Our data point to a potential metabolic link between accumulations of tyrosine and seeding of pathological aggregates in neurons as initiators for the pathological processes involved in neurodegeneration. Hence, interventions in tyrosine metabolism via regulation of NO levels may be therapeutic beneficial for the treatment of catecholamine-related neurodegenerative disorders.

Argininosuccinate Lyase Deficiency Causes an Endothelial-Dependent Form of Hypertension.

Primary hypertension is a major risk factor for ischemic heart disease, stroke, and chronic kidney disease. Insights obtained from the study of rare Mendelian forms of hypertension have been invaluable in elucidating the mechanisms causing primary hypertension and development of antihypertensive therapies. Endothelial cells play a key role in the regulation of blood pressure; however, a Mendelian form of hypertension that is primarily due to endothelial dysfunction has not yet been described. Here, we show that the urea cycle disorder, argininosuccinate lyase deficiency (ASLD), can manifest as a Mendelian form of endothelial-dependent hypertension. Using data from a human clinical study, a mouse model with endothelial-specific deletion of argininosuccinate lyase (Asl), and in vitro studies in human aortic endothelial cells and induced pluripotent stem cell-derived endothelial cells from individuals with ASLD, we show that loss of ASL in endothelial cells leads to endothelial-dependent vascular dysfunction with reduced nitric oxide (NO) production, increased oxidative stress, and impaired angiogenesis. Our findings show that ASLD is a unique model for studying NO-dependent endothelial dysfunction in human hypertension.

Biomarkers for liver disease in urea cycle disorders.

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

Variants associated with urea cycle disorders in Japanese patients: Nationwide study and literature review.

Urea cycle disorders (UCDs) are inherited metabolic diseases that lead to hyperammonemia with variable clinical manifestations. Using data from a nationwide study, we investigated the onset time, gene variants, clinical manifestations, and treatment of patients with UCDs in Japan. Of the 229 patients with UCDs diagnosed and/or treated between January 2000 and March 2018, identified gene variants and clinical information were available for 102 patients, including 62 patients with ornithine transcarbamylase (OTC) deficiency, 18 patients with carbamoyl phosphate synthetase 1 (CPS1) deficiency, 16 patients with argininosuccinate synthetase (ASS) deficiency, and 6 patients with argininosuccinate lyase (ASL) deficiency. A total of 13, 10, 4, and 5 variants in the OTC, CPS1, ASS, and ASL genes were respectively identified as novel variants, which were neither registered in ClinVar databases nor previously reported. The onset time and severity in patients with UCD could be predicted based on the identified gene variants in each patient from this nationwide study and previous studies. This genetic information may help in predicting the long-term outcome and determining specific treatment strategies such as liver transplantation in patients with UCDs.

Arginine recycling in endothelial cells is regulated BY HSP90 and the ubiquitin proteasome system.

Despite the saturating concentrations of intracellular l-arginine, nitric oxide (NO) production in endothelial cells (EC) can be stimulated by exogenous arginine. This phenomenon, termed the "arginine paradox" led to the discovery of an arginine recycling pathway in which l-citrulline is recycled to l-arginine by utilizing two important urea cycle enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Prior work has shown that ASL is present in a NO synthetic complex containing hsp90 and endothelial NO synthase (eNOS). However, it is unclear whether hsp90 forms functional complexes with ASS and ASL and if it is involved regulating their activity. Thus, elucidating the role of hsp90 in the arginine recycling pathway was the goal of this study. Our data indicate that both ASS and ASL are chaperoned by hsp90. Inhibiting hsp90 activity with geldanamycin (GA), decreased the activity of both ASS and ASL and decreased cellular l-arginine levels in bovine aortic endothelial cells (BAEC). hsp90 inhibition led to a time-dependent decrease in ASS and ASL protein, despite no changes in mRNA levels. We further linked this protein loss to a proteasome dependent degradation of ASS and ASL via the E3 ubiquitin ligase, C-terminus of Hsc70-interacting protein (CHIP) and the heat shock protein, hsp70. Transient over-expression of CHIP was sufficient to stimulate ASS and ASL degradation while the over-expression of CHIP mutant proteins identified both TPR- and U-box-domain as essential for ASS and ASL degradation. This study provides a novel insight into the molecular regulation l-arginine recycling in EC and implicates the proteasome pathway as a possible therapeutic target to stimulate NO signaling.

Molecular characterization and ornithine-urea cycle genes expression in air-breathing magur catfish (Clarias magur) during exposure to high external ammonia.

The air-breathing magur catfish (Clarias magur) is a potential ureogenic teleost because of its functional ornithine-urea cycle (OUC), unlike typical freshwater teleosts. The ability to convert ammonia waste to urea was a significant step towards land-based life forms from aquatic predecessors. Here we investigated the molecular characterization of some OUC genes and the molecular basis of stimulation of ureogenesis via the OUC in magur catfish. The deduced amino acid sequences from the complete cDNA coding sequences of ornithine transcarbamyolase, argininosuccinate synthase, and argininosuccinate lyase indicated that phylogenetically magur catfish is very close to other ureogenic catfishes. Ammonia exposure led to a significant induction of major OUC genes and the gene products in hepatic and in certain non-hepatic tissues of magur catfish. Hence, it is reasonable to assume that the induction of ureogenesis in magur catfish under hyper-ammonia stress is mediated through the activation of OUC genes as an adaptational strategy.

From genotype to phenotype: Early prediction of disease severity in argininosuccinic aciduria.

Argininosuccinic aciduria (ASA) is an inherited urea cycle disorder and has a highly variable phenotypic spectrum ranging from individuals with lethal hyperammonemic encephalopathy, liver dysfunction, and cognitive deterioration, to individuals with a mild disease course. As it is difficult to predict the phenotypic severity, we aimed at identifying a reliable disease prediction model. We applied a biallelic expression system to assess the functional impact of pathogenic argininosuccinate lyase (ASL) variants and to determine the enzymatic activity of ASL in 58 individuals with ASA. This cohort represented 42 ASL gene variants and 42 combinations in total. Enzymatic ASL activity was compared with biochemical and clinical endpoints from the UCDC and E-IMD databases. Enzymatic ASL activity correlated with peak plasma ammonium concentration at initial presentation and with the number of hyperammonemic events (HAEs) per year of observation. Individuals with ≤9% of enzymatic activity had more severe initial decompensations and a higher annual frequency of HAEs than individuals above this threshold. Enzymatic ASL activity also correlated with the cognitive outcome and the severity of the liver disease, enabling a reliable severity prediction for individuals with ASA. Thus, enzymatic activity measured by this novel expression system can serve as an important marker of phenotypic severity.

Translational Detection of Nonproteinogenic Amino Acids Using an Engineered Complementary Cell-Free Protein Synthesis Assay.

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.

Rapid Disruption of Genes Specifically in Livers of Mice Using Multiplex CRISPR/Cas9 Editing.

BACKGROUND & AIMS: Despite advances in gene editing technologies, generation of tissue-specific knockout mice is time-consuming. We used CRISPR/Cas9-mediated genome editing to disrupt genes in livers of adult mice in just a few months, which we refer to as somatic liver knockouts. METHODS: In this system, Fah-/- mice are given hydrodynamic tail vein injections of plasmids carrying CRISPR/Cas9 designed to excise exons in Hpd; the Hpd-edited hepatocytes have a survival advantage in these mice. Plasmids that target Hpd and a separate gene of interest can therefore be used to rapidly generate mice with liver-specific deletion of nearly any gene product. RESULTS: We used this system to create mice with liver-specific knockout of argininosuccinate lyase, which develop hyperammonemia, observed in humans with mutations in this gene. We also created mice with liver-specific knockout of ATP binding cassette subfamily B member 11, which encodes the bile salt export pump. We found that these mice have a biochemical phenotype similar to that of Abcb11-/- mice. We then used this system to knock out expression of 5 different enzymes involved in drug metabolism within the same mouse. CONCLUSIONS: This approach might be used to develop new models of liver diseases and study liver functions of genes that are required during development.

Crystal structure and biochemical study on argininosuccinate lyase from Mycobacterium tuberculosis.

Argininosuccinate lyase (ASL) participates in arginine synthesis through catalysing a reversible reaction in which argininosuccinate (AS) converts into arginine and fumarate. ASL from Mycobacterium tuberculosis is essential for its growth. In this work, the crystal structure of the apo form of MtbASL was determined and reveals a tetrameric structure that is essential for its activity since the active sites are formed by residues from three different monomers. Subsequently, we determined the crystal structure of MtbASL-sulfate complex, and the ligand mimics the negatively charged intermediate. The complex structure and mutagenesis studies indicate that residues S282 and H161 might act as a catalytic dyad. A major conformational change in the MtbASL-SO4 complex structure could be observed upon sulfate binding, and this movement facilitates the interaction between substrate and the residues involved in catalysis. A different conformational change in the C-terminal domain could be observed in the MtbASL-SO4 complex compared with that in other homologues. This difference may be responsible for the lower activity of MtbASL, which is related to the slow growth rate of M. tuberculosis. The C-terminal domain is a potential allosteric site upon inhibitor binding. The various conformational changes and the diversity of the sequence of the potential allosteric site across the homologues might provide clues for designing selective inhibitors against M. tuberculosis.

KLF7 promotes polyamine biosynthesis and glioma development through transcriptionally activating ASL.

Krüppel-like factors (KLFs) are zinc-finger transcriptional factors that regulate target gene expression. Recent studies have shown that KLFs play essential roles in cancer development, whereas the function of KLF7 in glioma remains unclear. In this study, we showed that KLF7 was up-regulated in glioma tissues and its expression was inversely correlated with the patients' survival. Functional experiments demonstrated that KLF7 promoted the proliferation, migration and tumorigenesis of glioma cells. Mechanistically, KLF7 transcriptionally activated argininosuccinate lyase (ASL), which was observed highly expressed in glioma tissues. The biosynthesis of polyamine, a urea cycle metabolite, was enhanced by KLF7 in glioma cells. In addition, ASL contributed to the growth of glioma cells triggered by KLF7. Our findings demonstrate KLF7 as an oncogene and link KLF7 to ASL-mediated polyamine metabolism in glioma.

Structural studies on M. tuberculosis argininosuccinate lyase and its liganded complex: Insights into catalytic mechanism.

Argininosuccinate lyase catalyses the reversible breakdown of argininosuccinate into arginine and fumarate and is known to form tetramers in its quaternary association. The absence of structures involving competent enzymes bound to substrate/products came in the way of the precise elucidation of the catalytic mechanism of this family of proteins. Crystal structures of the enzyme from Mycobacterium tuberculosis in an unliganded form and its complex with the substrate/products have now been determined at 2.2 and 2.7 Å, respectively. The refinement of the structure of the complex was bedevilled by the presence of a lattice translocation defect. The two tetramers in the apo-crystals and the one in the crystals of the liganded protein, have the same structure except for the movements associated with enzyme action. Each molecule consists of an N-domain, an M-domain, and a C-domain. The molecule consists of four binding sites, each made up of peptide stretches from three subunits. Three binding sites appear to be occupied by the ligand in the transition state, while the products occupy the fourth site. The structure exhibits the movement of a loop in the M-domain and parts of the C-domain. This is the first instance when the appropriate movements are observed in a complex with bound substrate/product. The detailed picture of the binding site, active site residues and the movements associated with catalysis thus obtained, enabled a revisit of the mechanism of action of the enzyme. © 2019 IUBMB Life, 71(5):643-652, 2019.

A simple dual-inducible CRISPR interference system for multiple gene targeting in Corynebacterium glutamicum.

The development of CRISPR interference (CRISPRi) technology has dramatically increased the pace and the precision of target identification during platform strain development. In order to develop a simple, reliable, and dual-inducible CRISPRi system for the industrially relevant Corynebacterium glutamicum, we combined two different inducible repressor systems in a single plasmid to separately regulate the expression of dCas9 (anhydro-tetracycline-inducible) and a given single guide RNA (IPTG-inducible). The functionality of the resulting vector was demonstrated by targeting the l-arginine biosynthesis pathway in C. glutamicum. By co-expressing dCas9 and a specific single guide RNA targeting the 5'-region of the argininosuccinate lyase gene argH, the specific activity of the target enzyme was down-regulated and in a l-arginine production strain, l-arginine formation was shifted towards citrulline formation. The system was also employed for down-regulation of multiple genes by concatenating sgRNA sequences encoded on one plasmid. Simultaneous down-regulated expression of both argH and the phosphoglucose isomerase gene pgi proved the potential of the system for multiplex targeting. The system can be a promising tool for further pathway engineering in C. glutamicum. Cumulative effects on targeted genes can be rapidly evaluated avoiding tedious and time-consuming traditional gene knockout approaches.

Argininosuccinic aciduria: Recent pathophysiological insights and therapeutic prospects.

The first patients affected by argininosuccinic aciduria (ASA) were reported 60 years ago. The clinical presentation was initially described as similar to other urea cycle defects, but increasing evidence has shown overtime an atypical systemic phenotype with a paradoxical observation, that is, a higher rate of neurological complications contrasting with a lower rate of hyperammonaemic episodes. The disappointing long-term clinical outcomes of many of the patients have challenged the current standard of care and therapeutic strategy, which aims to normalize plasma ammonia and arginine levels. Interrogations have raised about the benefit of newborn screening or liver transplantation on the neurological phenotype. Over the last decade, novel discoveries enabled by the generation of new transgenic argininosuccinate lyase (ASL)-deficient mouse models have been achieved, such as, a better understanding of ASL and its close interaction with nitric oxide metabolism, ASL physiological role outside the liver, and the pathophysiological role of oxidative/nitrosative stress or excessive arginine treatment. Here, we present a collaborative review, which highlights these recent discoveries and novel emerging concepts about ASL role in human physiology, ASA clinical phenotype and geographic prevalence, limits of current standard of care and newborn screening, pathophysiology of the disease, and emerging novel therapies. We propose recommendations for monitoring of ASA patients. Ongoing research aims to better understand the underlying pathogenic mechanisms of the systemic disease to design novel therapies.

[Genetic diagnosis of a Chinese pedigree affected with neonatal argininosuccinic aciduria].

OBJECTIVE: To explore the genetic basis of a neonate with argininosuccinic aciduria (ASA). METHODS: A neonate with lethargy and food refusal was admitted. The patient had myoclonus, myasthenia, uroschesis, irregular breathing and paroxysmal ventricular tachycardia, and died at 75 hours after birth. Laboratory test showed marked increase in blood ammonia (1249.8 µmol/L). Peripheral blood samples of the patient, her parents and sister were collected and subjected to trio whole-exome sequencing. RESULTS: Whole-exome sequencing revealed that the patient has carried compound heterozygous mutations of the argininosuccinate lyase (ASL) gene, namely c.425(exon5)_c.426(exon5) insAGCTCCCAGCT (p.Thr142Thrfs*37) and c.626(exon8)delT (p.Leu209Argfs*42). The patient was diagnosed as ASA caused by ASL gene mutations. Her parents and her elder sister were heterozygous carriers of the above mutations and had a normal phenotype. CONCLUSION: ASA is a severe congenital genetic metabolic disease and can manifest as onset of hyperammonemia in neonates. The clinical diagnosis is difficult and ASL gene testing may be helpful.

Adeno-associated viral gene therapy corrects a mouse model of argininosuccinic aciduria.

Argininosuccinic aciduria (ASA) is the second most common genetic disorder affecting the urea cycle. The disease is caused by deleterious mutations in the gene encoding argininosuccinate lyase (ASL); total loss of ASL activity results in severe neonatal onset of the disease, which is characterized by hyperammonemia within a few days of birth that can rapidly progress to coma and death. The long-term complications of ASA, such as hypertension and neurocognitive deficits, appear to be resistant to the current treatment options of dietary restriction, arginine supplementation, and nitrogen scavenging drugs. Treatment-resistant disease is currently being managed by orthotopic liver transplant, which shows variable improvement and requires lifetime immunosuppression. Here, we developed a gene therapy strategy for ASA aimed at alleviating the symptoms associated with urea cycle disruption by providing stable expression of ASL protein in the liver. We designed a codon-optimized human ASL gene packaged within adeno-associated virus serotype 8 (AAV8) as a vector for targeted delivery to the liver. To evaluate the therapeutic efficacy of this approach, we utilized a murine hypomorphic model of ASA. Neonatal administration of AAV8 via the temporal facial vein extended survival in ASA hypomorphic mice, although not to wild-type levels. Intravenous injection into adolescent hypomorphic mice led to increased survival and body weight and correction of metabolites associated with the disease. Our results demonstrate that AAV8 gene therapy is a viable approach for the treatment of ASA.