RARE DISEASE
Citrullinemia type I
Citrullinemia type I
Citrullinemia type I
Synonyms: ASS deficiency, Argininosuccinate synthase deficiency, Argininosuccinate synthetase deficiency, Argininosuccinic acid synthase deficiency, Argininosuccinic acid synthetase deficiency, CTLN1, Citrullinemia type 1, Classic citrullinemia
Synonyms: ASS deficiency, Argininosuccinate synthase deficiency, Argininosuccinate synthetase deficiency, Argininosuccinic acid synthase deficiency, Argininosuccinic acid synthetase deficiency, CTLN1, Citrullinemia type 1, Classic citrullinemia
Synonyms: ASS deficiency, Argininosuccinate synthase deficiency, Argininosuccinate synthetase deficiency, Argininosuccinic acid synthase deficiency, Argininosuccinic acid synthetase deficiency, CTLN1, Citrullinemia type 1, Classic citrullinemia
Drug discovery
4
drugs
With orphan designations
Overview
Citrullinemia type I (CTLN1) is a rare autosomal recessive urea cycle disorder caused by ASS1 gene mutations, leading to argininosuccinate synthase deficiency. This results in impaired ammonia detoxification, causing hyperammonemia, elevated citrulline, and neurotoxic effects. Neonatal-onset presents with rapid deterioration (lethargy, seizures, coma), while late-onset variants manifest with episodic hyperammonemic crises. Diagnosis relies on newborn screening (elevated citrulline) and genetic testing. Management involves acute ammonia reduction (scavengers, dialysis) and chronic protein restriction with nitrogen-scavenging therapies [1][3][15].
Categories: rare genetic diseases, rare inborn errors of metabolism
Research Papers
124 drug discovery papers about Citrullinemia type I, with 3 first-in-class and 1 next-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
124 drug discovery papers about Citrullinemia type I, with 3 first-in-class and 1 next-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
categories:
Small molecules
gene therapies
2026-02-28 | Dual rare genetic variants: case report of a child with SBIDDS syndrome and citrullinemia type 1.
BACKGROUND: The arginine methyltransferase 7 (PRMT7) gene plays a role in signal transduction and protein interactions and negatively regulates neuronal differentiation. Pathogenic variants of PRMT7 cause SBIDDS syndrome (Short stature, brachydactyly, intellectual developmental disability, and seizures). PRMT7 has been shown to interact with the argininosuccinate synthetase (ASS1) gene; biallelic pathogenic variants of ASS1 are associated with citrullinemia type 1. We report a patient with biallelic variants for SBIDDS syndrome and citrullinemia type 1. CASE PRESENTATION: The child exhibits severe intellectual disability, microcephaly, dysmorphisms, and seizures, consistent with the characteristics of these conditions. Exome sequencing identified variants c.1575 +1 G > A and c.232 dupT;p.Leu78Phefs*24 (NM_019023) in compound heterozygosity in the PRMT7 gene and variants c.1168G > A;p.Gly390Arg and c.-5-10C > G in exon 3 of the ASS1 gene (previously also identified in neonatal metabolic screening). The patient also has Wolf-Parkinson-White syndrome (WPW), behavioral issues, and brain morphological abnormalities, features that are poorly described in prior studies. CONCLUSIONS: It is not yet clear whether the clinical severity observed in this patient could also be partially due to interactions between these variants, as such interactions are not well-documented in the literature. Further research is necessary to elucidate the interplay between these two genes.
2026-01-01 | Fatal Neonatal Citrullinemia Type I Unmasking a History of Recurrent Reproductive Loss in a Non-Consanguineous Indian Family: A Case Report
This case report describes a fatal neonatal presentation of Citrullinemia Type I (CTLN1) in a term male infant from a non-consanguineous Indian family, a diagnosis that eventually provided an explanation for a significant history of recurrent reproductive loss (G6P2A4L0). The neonate initially manifested with refractory hypoglycemia at 10 hours of life, necessitating glucose infusion rates up to 13~mg/kg/min$, before progressing to severe hyperammonemic encephalopathy with a peak ammonia level of 1280~\mu mol/L$. Despite intensive supportive care, the infant died on day 15; however, postmortem tandem mass spectrometry revealed a diagnostic citrulline elevation of 1100~\mu mol/L, and whole exome sequencing identified a homozygous pathogenic splice-site variant in the ASS1 gene (c.421-2A>G). The case underscores the critical need for expedited newborn screening infrastructure in India, as the 20-day turnaround for biochemical results arrived only after the patient’s death, while highlighting how community endogamy can mimic consanguinity to facilitate rare autosomal recessive disorders
2025-08-21 | Case Report: From coma to genetic insights: identification of a novel pathogenic variant in Chinese neonatal CTLN1.
Citrullinemia type I (CTLN1) is an autosomal recessive disorder caused by variants in the arginine succinate synthase gene (ASS1). These variants result in arginine succinate synthase deficiency, leading to a disruption of the urea cycle and hyperammonemia. To date, only a handful of CTLN1 cases have been reported in China. One neonate responded poorly 30 h after birth and progressed to coma several hours later. Family history revealed that the neonate's older brother had also died a few days after birth. Biochemical tests on admission confirmed hyperammonemia and elevated levels of citrulline and urinary orotic acid-3. Genetic analysis revealed that the parents were carriers of two heterozygous variants in ASS1, c.910C>T(p.Arg304Trp) and c.839-1G>A, respectively. However, the splice site variant c.839-1G>A was not present in the control databases. Minigene analysis of the c.839-1G>A resulted in the product of r.839del [p.(Gly280Valfs*15)]. In conclusion, we have identified a case of CTLN1 and diagnosed a novel pathogenic variant in the ASS1 gene, c.839-1G>A, expanding the variant spectrum of ASS1. Currently, there are few reports of CTLN1 cases featuring such severe clinical manifestations and an onset at such a young age.
2025-08-01 | BLOOD AMMONIA AS EARLY PREDICTOR OF CITRULLINEMIA TYPE 1 IN NEW BORN – A RARE CASE REPORT
Citrullinemia type 1 is an autosomal recessive urea cycle disorder that causes ammonia and other toxic substances to accumulate in the blood.We are reporting a rare case of citrullinemia Type 1 in a female Term neonate.The baby was brought to our Neonatal Intensive Care Unit on day 5 with complaints of lethargy and poor feeding.Initial blood investigations showed elevated blood ammonia and low urea levels, suggesting an Inborn Error of metabolism (IEM).Blood Tandem Mass spectrometry (TMS) and Urine Gas Chromatography Mass Spectrometry (GC-MS)
2024-03-27 | Regulation of urea cycle by reversible high-stoichiometry lysine succinylation.
The post-translational modification lysine succinylation is implicated in the regulation of various metabolic pathways. However, its biological relevance remains uncertain due to methodological difficulties in determining high-impact succinylation sites. Here, using stable isotope labelling and data-independent acquisition mass spectrometry, we quantified lysine succinylation stoichiometries in mouse livers. Despite the low overall stoichiometry of lysine succinylation, several high-stoichiometry sites were identified, especially upon deletion of the desuccinylase SIRT5. In particular, multiple high-stoichiometry lysine sites identified in argininosuccinate synthase (ASS1), a key enzyme in the urea cycle, are regulated by SIRT5. Mutation of the high-stoichiometry lysine in ASS1 to succinyl-mimetic glutamic acid significantly decreased its enzymatic activity. Metabolomics profiling confirms that SIRT5 deficiency decreases urea cycle activity in liver. Importantly, SIRT5 deficiency compromises ammonia tolerance, which can be reversed by the overexpression of wild-type, but not succinyl-mimetic, ASS1. Therefore, lysine succinylation is functionally important in ammonia metabolism.
proteins
2025-09-20 | Functional Profiling of 2,193 ASS1 Missense Variants: Insights into Variant Pathogenicity and Epistatic Interactions in Citrullinemia Type I
Sequence variants in the urea cycle gene argininosuccinate synthase (ASS1) cause Citrullinemia type 1 (CTLN1), a rare autosomal recessive disease. Mechanistically, reduction in argininosuccinate synthetase (ASS) enzyme activity impairs the urea cycle, leading to an accumulation of citrulline and neurotoxic ammonia. Disease severity varies according to the degree of enzyme impairment, ranging from severe neonatal forms (classic citrullinemia) to milder, late-onset forms that may manifest in childhood or adulthood. We established a high-throughput yeast functional assay of human ASS and individually measured the impact of 2193 amino acid substitutions, representing 90% of all single nucleotide variant (SNV)-accessible substitutions. When benchmarked against existing clinical variant annotation, our assay distinguishes known benign variants from strong loss of function pathogenic variants, enabling identification of a functional score threshold below which variants show clinically relevant impairment of ASS activity. Using the ACMG OddsPath framework, our assay meets PS3_supporting criteria for pathogenicity classification and achieves full PS3-level strength when variants observed as homozygotes in other primates are used as benign proxies for calibration. These results provide direct functional evidence to inform reclassification of ASS1 missense variants, supporting their clinical interpretation and diagnostic utility. Mapping functional scores onto the protein structure, we confirmed that residues involved in catalysis are highly sensitive to substitution. In addition, we identified residues from adjacent subunits of the ASS homotetramer that form compound active sites. Assaying these positions revealed a capacity for intragenic complementation consistent with a variant sequestration model: a form of positive epistasis in which deleterious variants from different subunits are sequestered into only a subset of active sites, restoring function in the remaining variant-free sites. The discovery of intragenic complementation in ASS reveals a novel mode of functional interaction with clinical implications for interpreting variant combinations in heterozygous individuals.
2022-03-15 | Phase 1 trial of ADI-PEG 20 and liposomal doxorubicin in patients with metastatic solid tumors.
Arginine depletion interferes with pyrimidine metabolism and DNA damage repair pathways. Preclinical data demonstrated that depletion of arginine by PEGylated arginine deiminase (ADI-PEG 20) enhanced liposomal doxorubicin (PLD) cytotoxicity in cancer cells with argininosuccinate synthase 1 (ASS1) deficiency. The objective of this study was to assess safety and tolerability of ADI-PEG 20 and PLD in patients with metastatic solid tumors. Patients with advanced ASS1-deficient solid tumors were enrolled in this phase 1 trial of ADI-PEG 20 and PLD following a 3 + 3 design. Eligible patients were given intravenous PLD biweekly and intramuscular (IM) ADI-PEG 20 weekly. Toxicity and efficacy were evaluated according to the Common Terminology Criteria for Adverse Events (version 4.0) and Response Evaluation Criteria in Solid Tumors (version 1.1), respectively. Of 15 enrolled patients, 9 had metastatic HER2-negative breast carcinoma. We observed no dose-limiting toxicities or treatment-related deaths. One patient safely received 880 mg/m2 PLD in this study and 240 mg/m2 doxorubicin previously. Treatment led to stable disease in 9 patients and was associated with a median progression-free survival time of 3.95 months in 15 patients. Throughout the duration of treatment, decreased arginine and increased citrulline levels in peripheral blood remained significant in a majority of patients. We detected no induction of anti-ADI-PEG 20 antibodies by week 8 in one third of patients. Concurrent IM injection of ADI-PEG 20 at 36 mg/m2 weekly and intravenous infusion of PLD at 20 mg/m2 biweekly had an acceptable safety profile in patients with advanced ASS1-deficient solid tumors. Further evaluation of this combination is under discussion.
2021-03-05 | Phase 1 trial of ADI-PEG20 plus cisplatin in patients with pretreated metastatic melanoma or other advanced solid malignancies
Abstract Background Arginine depletion interferes with pyrimidine metabolism and DNA damage-repair pathways, and pairing arginine deiminase pegylated with 20,000-molecular-weight polyethylene glycol (ADI-PEG20) with platinum enhances cytotoxicity in vitro and in vivo in arginine auxotrophs. Methods This single-centre, Phase 1 trial was conducted using a 3 + 3 dose escalation designed to assess safety, tolerability and determine the recommended Phase 2 dose (RP2D) of ADI-PEG20. Results We enrolled 99 patients with metastatic argininosuccinate synthetase 1 (ASS1) deficient malignancies. We observed no dose-limiting toxic effects or treatment-related mortality. Three percent of patients discontinued treatment because of toxicity. After treatment, 5% (5/99) of patients had partial responses, and 41% had stable disease. The median progression-free and overall survival durations were 3.62 and 8.06 months, respectively. Substantial arginine depletion and citrulline escalation persisted in most patients through weeks 24 and 8, respectively. Tumour responses were associated with anti-ADI-PEG20 antibody levels at weeks 8 and 16 ( p = 0.031 and p = 0.0357, respectively). Conclusion Concurrently administered ADI-PEG20 and cisplatin had an acceptable safety profile and had shown antitumour activity against metastatic ASS1-deficient solid tumours. Further evaluation of this treatment combination is warranted.
2020-06-19 | A Phase I Study of Pegylated Arginine Deiminase (Pegargiminase), Cisplatin, and Pemetrexed in Argininosuccinate Synthetase 1-Deficient Recurrent High-grade Glioma.
Patients with recurrent high-grade gliomas (HGG) are usually managed with alkylating chemotherapy ± bevacizumab. However, prognosis remains very poor. Preclinically, we showed that HGGs are a target for arginine depletion with pegargiminase (ADI-PEG20) due to epimutations of argininosuccinate synthetase (ASS1) and/or argininosuccinate lyase (ASL). Moreover, ADI-PEG20 disrupts pyrimidine pools in ASS1-deficient HGGs, thereby impacting sensitivity to the antifolate, pemetrexed. We expanded a phase I trial of ADI-PEG20 with pemetrexed and cisplatin (ADIPEMCIS) to patients with ASS1-deficient recurrent HGGs (NCT02029690). Patients were enrolled (01/16-06/17) to receive weekly ADI-PEG20 36 mg/m2 intramuscularly plus pemetrexed 500 mg/m2 and cisplatin 75 mg/m2 intravenously once every 3 weeks for up to 6 cycles. Patients with disease control were allowed ADI-PEG20 maintenance. The primary endpoints were safety, tolerability, and preliminary estimates of efficacy. Ten ASS1-deficient heavily pretreated patients were treated with ADIPEMCIS therapy. Treatment was well tolerated with the majority of adverse events being Common Terminology Criteria for Adverse Events v4.03 grade 1-2. The best overall response was stable disease in 8 patients (80%). Plasma arginine was suppressed significantly below baseline with a reciprocal increase in citrulline during the sampling period. The anti-ADI-PEG20 antibody titer rose during the first 4 weeks of treatment before reaching a plateau. Median progression-free survival (PFS) was 5.2 months (95% confidence interval (CI), 2.5-20.8) and overall survival was 6.3 months (95% CI, 1.8-9.7). In this recurrent HGG study, ADIPEMCIS was well tolerated and compares favorably to historical controls. Additional trials of ADI-PEG20 in HGG are planned.
2015-08-26 | Argininosuccinate synthetase (ASS) deficiency in high-grade pulmonary neuroendocrine carcinoma: an opportunity for personalized targeted therapy.
Cells deficient in argininosuccinate synthetase (ASS) must absorb the arginine they need for growth from circulating blood. Treatment with pegylated arginine deiminase (ADI-PEG 20) selectively eliminates arginine from the circulation and has shown some efficacy against ASS-deficient tumors including small cell lung cancer (SCLC). We sought to assess ASS expression in a cohort of high-grade pulmonary neuroendocrine carcinomas (PNEC) which include SCLC and large cell neuroendocrine carcinoma (LCNEC). Sixty-nine PNEC (49 SCLC and 20 LCNEC) were retrieved from our pathology archives. Formalin-fixed paraffin-embedded sections of the 54 primary tumors, 15 metastases and appropriate positive and negative controls were immunostained using an ASS-specific monoclonal antibody. Positive staining in <30 % of the tumor was scored as weak; staining in ≥30 % of the tumor was scored as strong. The absence of staining in the tumor was recorded as ASS negative. 58 % of the PNEC including 61.2 % of the SCLC and 50 % of the LCNEC were ASS negative. These ASS-negative tumors included 63 % of the primary and 40 % of the metastatic lesions tested. More than 50 % of the high-grade PNEC tested lack immunohistochemically detectable ASS, suggesting that they are auxotrophic for arginine and potential candidates for arginine deprivation therapy. PNEC comprise about 25 % of primary lung cancers and have a 5-year overall survival of only 5-10 %, underscoring the need for new and more effective therapies. Immunostaining for ASS has potential to improve the selection of patients with PNEC for arginine deprivation therapy with ADI-PEG 20.
oligonucleotides
2026-05-13 | RNA-LNP-mediated in vivo prime editing corrects disease phenotypes in a mouse model of citrullinemia type I.
Citrullinemia type I (CTLN1) is a severe urea cycle disorder caused by pathogenic variants in the ASS1 (argininosuccinate synthetase 1) gene, for which liver transplantation remains the only curative option. Here, we used prime editing to correct the Ass1fold mouse model of CTLN1. Adeno-associated virus (AAV)-mediated delivery of the PE7 prime editor with an optimized prime editing guide RNA (pegRNA) achieved 71 and 54% correction of the pathogenic Ass1 mutation in hepatocytes of neonates and juveniles, respectively. Delivery of mRNA-encoded PE7 and synthetic pegRNA via lipid nanoparticles (LNPs) resulted in 24% correction after a single 3 mg kg-1 dose in neonates and 13% after three 4 mg kg-1 doses in juveniles. All treated groups showed full normalization of survival and of blood citrulline and ammonia concentrations, with restored urea cycle function and correction of natural behavior defects. Consistent with these findings, immunostaining demonstrated restoration of wild-type-like ASS1 protein localization in functionally relevant periportal and intermediate-zone hepatocytes. Editing was confined to the liver, with minimal indel formation and off-target activity and only transient elevations in liver enzymes. In a cellular reporter system, 6 of 15 recurrent human pathogenic ASS1 mutations studied, including the most common ASS1G390R variant, were corrected with similar or higher efficiencies than Ass1fold. These findings highlight prime editing as a precise and potentially curative treatment strategy for individuals with CTLN1 and other genetic liver diseases.
2024-10-25 | Exploring RNA therapeutics for urea cycle disorders.
RNA has triggered a significant shift in modern medicine, providing a promising way to revolutionize disease treatment methods. Different therapeutic RNA modalities have shown promise to replace, supplement, correct, suppress, or eliminate the expression of a targeted gene. Currently, there are 22 RNA-based drugs approved for clinical use, including the COVID-19 mRNA vaccines, whose unprecedented worldwide success has meant a definitive boost in the RNA research field. Urea cycle disorders (UCD), liver diseases with high mortality and morbidity, may benefit from the progress achieved, as different genetic payloads have been successfully targeted to liver using viral vectors, N-acetylgalactosamine (GalNAc) conjugations or lipid nanoparticles (LNP). This review explores the potential of RNA-based medicines for UCD and the ongoing development of applications targeting specific gene defects, enzymes, or transporters taking part in the urea cycle. Notably, LNP-formulated mRNA therapy has been assayed preclinically for citrullinemia type I (CTLN1), adolescent and adult citrin deficiency, argininosuccinic aciduria, arginase deficiency and ornithine transcarbamylase deficiency, in the latter case has progressed to the clinical trials phase.
2024-01-22 | ASS1 deficiency is associated with impaired neuronal differentiation in zebrafish larvae.
Citrullinemia type 1 (CTLN1) is a rare autosomal recessive urea cycle disorder caused by deficiency of the cytosolic enzyme argininosuccinate synthetase 1 (ASS1) due to pathogenic variants in the ASS1 gene located on chromosome 9q34.11. Even though hyperammenomia is considered the major pathomechanistic factor for neurological impairment and cognitive dysfunction, a relevant subset of individuals presents with a neurodegenerative course in the absence of hyperammonemic decompensations. Here we show, that ASS1 deficiency induced by antisense-mediated knockdown of the zebrafish ASS1 homologue is associated with defective neuronal differentiation ultimately causing neuronal cell loss and consecutively decreased brain size in zebrafish larvae in vivo. Whereas ASS1-deficient zebrafish larvae are characterized by markedly elevated concentrations of citrulline - the biochemical hallmark of CTLN1, accumulation of L-citrulline, hyperammonemia or therewith associated secondary metabolic alterations did not account for the observed phenotype. Intriguingly, coinjection of the human ASS1 mRNA not only normalized citrulline concentration but also reversed the morphological cerebral phenotype and restored brain size, confirming conserved functional properties of ASS1 across species. The results of the present study imply a novel, potentially non-enzymatic (moonlighting) function of the ASS1 protein in neurodevelopment.
2024-01-10 | mRNA therapy corrects defective glutathione metabolism and restores ureagenesis in preclinical argininosuccinic aciduria
The urea cycle enzyme argininosuccinate lyase (ASL) enables the clearance of neurotoxic ammonia and the biosynthesis of arginine. Patients with ASL deficiency present with argininosuccinic aciduria, an inherited metabolic disease with hyperammonemia and a systemic phenotype coinciding with neurocognitive impairment and chronic liver disease. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics and in vivo positron emission tomography (PET) imaging using ( S )-4-(3- 18 F-fluoropropyl)- l -glutamate ([ 18 F]FSPG). Up-regulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways. To assess hepatic glutathione dysregulation and liver disease, we present [ 18 F]FSPG PET as a noninvasive diagnostic tool to monitor therapeutic response in argininosuccinic aciduria. Human hASL mRNA encapsulated in lipid nanoparticles improved glutathione metabolism and chronic liver disease. In addition, hASL mRNA therapy corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis. These findings provide mechanistic insights in liver glutathione metabolism and support clinical translation of mRNA therapy for argininosuccinic aciduria.
2023-12-14 | Ex vivo precision-cut liver slices model disease phenotype and monitor therapeutic response for liver monogenic diseases
Background In academic research and the pharmaceutical industry, in vitro cell lines and in vivo animal models are considered as gold standards in modelling diseases and assessing therapeutic efficacy. However, both models have intrinsic limitations, whilst the use of precision-cut tissue slices can bridge the gap between these mainstream models. Precision-cut tissue slices combine the advantage of high reproducibility, studying all cell sub-types whilst preserving the tissue matrix and extracellular architecture, thereby closely mimicking a mini-organ. This approach can be used to replicate the biological phenotype of liver monogenic diseases using mouse models. Methods Here, we describe an optimised and easy-to-implement protocol for the culture of sections from mouse livers, enabling its use as a reliable ex-vivo model to assess the therapeutic screening of inherited metabolic diseases Results We show that precision-cut liver sections can be a reliable model for recapitulating the biological phenotype of inherited metabolic diseases, exemplified by common urea cycle defects such as citrullinemia type 1 and argininosuccinic aciduria, caused by argininosuccinic synthase (ASS1) and argininosuccinic lyase (ASL) deficiencies respectively. Conclusions Therapeutic response to gene therapy such as messenger RNA replacement delivered via lipid nanoparticles can be monitored, demonstrating that precision-cut liver sections can be used as a preclinical screening tool to assess therapeutic response and toxicity in monogenic liver diseases.
small molecules
2026-01-01 | P047: Incidental identification of citrullinemia type 1 through carrier screening post liver transplant
Outcome and Follow-Up: Following the addition of carglumic acid, we were able to gradually wean our patient off all additional urea cycle disorder (UCD) management -ammonia scavengers and medical formula -over the course of five months.The patient has not experienced any further hyperammonemic episodes.Citrulline supplementation was discontinued at 11 months and plasma citrulline levels have remained normal to date.Glutamine and alanine levels have remained in normal range, with a maximum glutamine level of 671mol/L.At 21 months of age, the patient remains metabolically stable on carglumic acid 53.6mg/kg/day with dietary protein allotment not exceeding 2.2g/kg/day from intact protein without UCD formula.Developmentally he demonstrates mild-to-moderate delays; likely reflecting his hyperammonemia at presentation.Discussion: This case highlights the importance of early recognition and prompt initiation of carglumic acid in this diagnosis.The identified NAGS c.1289T>C variant adds to the catalog of pathogenic variants identified in this gene.Carglumic acid, an analog of N-acetylglutamate, has proven an effective sole treatment for this enzyme deficiency in our patient with sustained metabolic control.Conclusion: Early diagnosis and prompt initiation of carglumic acid therapy are lifesaving and life-altering in NAGS deficiency.This case contributes to the very limited clinical literature about this diagnosis and its long-term management, while adding to the clinical experience a novel variant.
2025-02-03 | BCL-XL Protects ASS1-Deficient Cancers from Arginine Starvation-Induced Apoptosis.
Argininosuccinate synthetase 1 (ASS1) silencing in carcinomas and sarcomas leads to a dependence on extracellular arginine for survival. Arginine deprivation therapies, such as PEGylated arginine deiminase (ADI-PEG20), have shown limited effectiveness, which may be due to underlying mechanisms that inhibit apoptosis. The effects of ADI-PEG20 on cell-cycle regulation, apoptosis, and BCL-XL-mediated survival pathways in ASS1-deficient cancer cells were determined. The mechanism of cell death protection was determined by assessing caspase and PARP cleavage, CDK2 activity, MCL1 expression, and the interactions among BCL-XL, BAX, and BAK. In vitro synergy was determined, and in vivo efficacy was modeled. Treatment with ADI-PEG20 led to reduced CDK2 activity and inhibited cell-cycle progression but did not induce significant cell death. BCL-XL was found to bind to BAX and BAK, preventing the initiation of apoptosis despite arginine starvation. Inhibition of BCL-XL allowed proapoptotic BAX and BAK to initiate the intrinsic apoptosis pathway, leading to increased cell death. This was found to be synergistic in vitro and efficacious in combination in vivo. The study identifies BCL-XL as a key factor limiting the efficacy of arginine starvation therapies. Combining BCL-XL inhibitors with arginine deprivation strategies may overcome this resistance and enhance therapeutic outcomes. These findings provide a strong preclinical rationale for testing this combination approach in phase 1 clinical trials for ASS1-deficient cancers.
2024-09-16 | Data from Discovery and targeting of a noncanonical mechanism of sarcoma resistance to ADI-PEG20 mediated by the microenvironment
<div>Abstract<p>Purpose: Many cancers lack argininosuccinate synthetase 1 (ASS1), the rate limiting enzyme of arginine biosynthesis. This deficiency causes arginine auxotrophy, targetable by extracellular arginine-degrading enzymes such as ADI-PEG20. Long-term tumor resistance has thus far been attributed solely to ASS1 re-expression. This study examines the role of ASS1 silencing on tumor growth and initiation and identifies a noncanonical mechanism of resistance, aiming to improve clinical responses to ADI-PEG20. Experimental Design: Tumor initiation and growth rates were measured for a spontaneous <i>Ass1</i> knockout (KO) murine sarcoma model. Tumor cell lines were generated, and resistance to arginine deprivation therapy was studied <i>in vitro </i>and<i> in vivo</i>. Results: Conditional <i>Ass1</i> KO affected neither tumor initiation nor growth rates in a sarcoma model, contradicting the prevalent idea that ASS1 silencing confers a proliferative advantage. <i>Ass1</i> KO cells grew robustly through arginine starvation <i>in vivo, </i>while ADI-PEG20 remained completely lethal <i>in vitro</i>, evidence that pointed toward a novel mechanism of resistance mediated by the microenvironment. Co-culture with <i>Ass1</i>-competent fibroblasts rescued growth through macropinocytosis of vesicles and/or cell fragments, followed by recycling of protein-bound arginine through autophagy/lysosomal degradation. Inhibition of either macropinocytosis or autophagy/lysosomal degradation abrogated this growth support effect <i>in vitro</i> and <i>in vivo</i>. Conclusions: Noncanonical, ASS1-independent tumor resistance to ADI-PEG20 is driven by the microenvironment. This mechanism can be targeted by either the macropinocytosis inhibitor imipramine or the autophagy inhibitor chloroquine. These safe, widely available drugs should be added to current clinical trials to overcome microenvironmental arginine support of tumors and improve patient outcomes.</p></div>
2023-11-09 | Case report: Diagnosis of ADCY5-related dyskinesia explaining the entire phenotype in a patient with atypical citrullinemia type I
In this case study, we report the case of a 13-year-old girl with citrullinemia type 1 (MIM #215700), an autosomal recessive inherited disorder of the urea cycle, which was confirmed by the identification of a homozygous pathogenic variant in the argininosuccinate synthetase 1 (ASS1) gene. However, the patient presented abnormal hyperkinetic movements with global developmental delay and clinical signs that were not fully consistent with those of citrullinemia type 1 or with those of her siblings with isolated citrullinemia type 1. Exome sequencing showed the presence of a de novo heterozygous pathogenic variant in the adenylate cyclase type 5 (ADCY5) gene. The variant confirmed the overlap with the so-called ADCY5-related dyskinesia with orofacial involvement, which is autosomal dominant (MIM #606703), a disorder disrupting the enzymatic conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). In addition to the citrullinemia-related low-protein diet and arginine supplementation, the identification of this second disease led to the introduction of a treatment with caffeine, which considerably improved the dyskinesia neurological picture. In conclusion, this case highlights the importance of clinical-biological confrontation for the interpretation of genetic variants, as one hereditary metabolic disease may hide another with therapeutic consequences.This article reports the misleading superposition of two inherited metabolic diseases, showing the importance of clinical-biological confrontation in the interpretation of genetic variants.
2023-09-01 | Evaluation of the Neurodevelopmental Status for Urea Cycle Disorders: Based on Clinical Experience
Aim: Urea cycle disorders (UCD) still have poor neurological outcomes despite early diagnosis and treatment.We aimed to present the neurological outcomes of UCD patients and to determine the main simple and accessible factors affecting these outcomes. Materials and Methods:This was a descriptive cross-sectional study conducted in two pediatric metabolism centers on 29 patients from 25 unrelated families who were diagnosed and followed with UCD based on clinical presentation, neurological parameters, biochemical measurements, and molecular analysis.Results: Within the study population, the most common diagnosis was argininosuccinate synthase deficiency in 13 (44.82%)patients, followed by N-acetylglutamate synthase deficiency in five patients (17.24%), ornithine transcarbamylase deficiency in four patients (13.79%), arginase 1 deficiency in three patients (10.34%), carbamoyl phosphate synthase 1 deficiency in three patients (10.34%), and argininosuccinate lyase deficiency in one patient (3.44%).Peak ammonia levels were observed to be significantly higher in those patients with delayed milestones and patients who had Denver II <-2 standard deviation score results (p=0.032,p=0.026).Effect sizes were large in both groups.Delayed milestones were noted in 17 (94.4%) of the cases with peak ammonia >500 μmol/L (n=18).Those patients with abnormal neurological parameters had a significantly higher mean number of hyperammonemic episodes per year.Extracorporeal detoxification was given to eight patients, in combination with therapeutic hypothermia in two patients.Rapid regression was observed in brain edema in those who underwent therapeutic hypothermia. Conclusion:Our study emphasizes the effect of peak ammonia levels and the frequency of hyperammonemic episodes on neurological outcomes.There were still poor neurocognitive outcomes despite extracorporeal detoxification.This highlights the need to reassess current treatment strategies, including the threshold for starting extracorporeal detoxification if ammonia levels exceed 500 μmol/L.The use of therapeutic hypothermia by experienced teams may be promising due to its brain edema-reducing effects.
other
2022-11-16 | Managing recurrent portal steal in auxiliary liver transplantation for non-cirrhotic metabolic liver disease.
APOLT has been proposed as a treatment modality for certain types of NCMLD. While the short-term outcomes of this operation have been comparable with orthotopic LT, its long-term outcomes have sparsely been reported. We present one such case of Citrullinemia type I who underwent APOLT and developed recurrent PS. A 2-year-old male child with a diagnosis of Citrullinemia type I underwent APOLT with a left lateral segment from a split deceased donor liver, and his postoperative period was unremarkable. Ammonia-lowering agents were stopped 1 week following the operation and the child was discharged home on a normal diet. Four years following APOLT, the child presented with altered sensorium and seizures. A diagnosis of PS was made. Subsequent to an embolization of the native liver's right anterior portal vein his sensorium improved and he remained clinically stable on a normal diet. Six years following the APOLT, the child again presented with features of acute encephalopathy. Imaging was suggestive of PS. A portal vein embolization of the native portal vein was performed and the child's clinical condition improved. At 6 months' follow-up, the child remains well on a normal diet. While the early impediments in this technique may have been overcome, in the absence of any realistic clinical application gene therapy, the debate of long-term phenotypic metabolic correction for NCMLD by APOLT needs to be revisited.
gene therapies
2026-02-28 | Dual rare genetic variants: case report of a child with SBIDDS syndrome and citrullinemia type 1.
BACKGROUND: The arginine methyltransferase 7 (PRMT7) gene plays a role in signal transduction and protein interactions and negatively regulates neuronal differentiation. Pathogenic variants of PRMT7 cause SBIDDS syndrome (Short stature, brachydactyly, intellectual developmental disability, and seizures). PRMT7 has been shown to interact with the argininosuccinate synthetase (ASS1) gene; biallelic pathogenic variants of ASS1 are associated with citrullinemia type 1. We report a patient with biallelic variants for SBIDDS syndrome and citrullinemia type 1. CASE PRESENTATION: The child exhibits severe intellectual disability, microcephaly, dysmorphisms, and seizures, consistent with the characteristics of these conditions. Exome sequencing identified variants c.1575 +1 G > A and c.232 dupT;p.Leu78Phefs*24 (NM_019023) in compound heterozygosity in the PRMT7 gene and variants c.1168G > A;p.Gly390Arg and c.-5-10C > G in exon 3 of the ASS1 gene (previously also identified in neonatal metabolic screening). The patient also has Wolf-Parkinson-White syndrome (WPW), behavioral issues, and brain morphological abnormalities, features that are poorly described in prior studies. CONCLUSIONS: It is not yet clear whether the clinical severity observed in this patient could also be partially due to interactions between these variants, as such interactions are not well-documented in the literature. Further research is necessary to elucidate the interplay between these two genes.
2026-01-01 | Fatal Neonatal Citrullinemia Type I Unmasking a History of Recurrent Reproductive Loss in a Non-Consanguineous Indian Family: A Case Report
This case report describes a fatal neonatal presentation of Citrullinemia Type I (CTLN1) in a term male infant from a non-consanguineous Indian family, a diagnosis that eventually provided an explanation for a significant history of recurrent reproductive loss (G6P2A4L0). The neonate initially manifested with refractory hypoglycemia at 10 hours of life, necessitating glucose infusion rates up to 13~mg/kg/min$, before progressing to severe hyperammonemic encephalopathy with a peak ammonia level of 1280~\mu mol/L$. Despite intensive supportive care, the infant died on day 15; however, postmortem tandem mass spectrometry revealed a diagnostic citrulline elevation of 1100~\mu mol/L, and whole exome sequencing identified a homozygous pathogenic splice-site variant in the ASS1 gene (c.421-2A>G). The case underscores the critical need for expedited newborn screening infrastructure in India, as the 20-day turnaround for biochemical results arrived only after the patient’s death, while highlighting how community endogamy can mimic consanguinity to facilitate rare autosomal recessive disorders
2025-08-21 | Case Report: From coma to genetic insights: identification of a novel pathogenic variant in Chinese neonatal CTLN1.
Citrullinemia type I (CTLN1) is an autosomal recessive disorder caused by variants in the arginine succinate synthase gene (ASS1). These variants result in arginine succinate synthase deficiency, leading to a disruption of the urea cycle and hyperammonemia. To date, only a handful of CTLN1 cases have been reported in China. One neonate responded poorly 30 h after birth and progressed to coma several hours later. Family history revealed that the neonate's older brother had also died a few days after birth. Biochemical tests on admission confirmed hyperammonemia and elevated levels of citrulline and urinary orotic acid-3. Genetic analysis revealed that the parents were carriers of two heterozygous variants in ASS1, c.910C>T(p.Arg304Trp) and c.839-1G>A, respectively. However, the splice site variant c.839-1G>A was not present in the control databases. Minigene analysis of the c.839-1G>A resulted in the product of r.839del [p.(Gly280Valfs*15)]. In conclusion, we have identified a case of CTLN1 and diagnosed a novel pathogenic variant in the ASS1 gene, c.839-1G>A, expanding the variant spectrum of ASS1. Currently, there are few reports of CTLN1 cases featuring such severe clinical manifestations and an onset at such a young age.
2025-08-01 | BLOOD AMMONIA AS EARLY PREDICTOR OF CITRULLINEMIA TYPE 1 IN NEW BORN – A RARE CASE REPORT
Citrullinemia type 1 is an autosomal recessive urea cycle disorder that causes ammonia and other toxic substances to accumulate in the blood.We are reporting a rare case of citrullinemia Type 1 in a female Term neonate.The baby was brought to our Neonatal Intensive Care Unit on day 5 with complaints of lethargy and poor feeding.Initial blood investigations showed elevated blood ammonia and low urea levels, suggesting an Inborn Error of metabolism (IEM).Blood Tandem Mass spectrometry (TMS) and Urine Gas Chromatography Mass Spectrometry (GC-MS)
2024-03-27 | Regulation of urea cycle by reversible high-stoichiometry lysine succinylation.
The post-translational modification lysine succinylation is implicated in the regulation of various metabolic pathways. However, its biological relevance remains uncertain due to methodological difficulties in determining high-impact succinylation sites. Here, using stable isotope labelling and data-independent acquisition mass spectrometry, we quantified lysine succinylation stoichiometries in mouse livers. Despite the low overall stoichiometry of lysine succinylation, several high-stoichiometry sites were identified, especially upon deletion of the desuccinylase SIRT5. In particular, multiple high-stoichiometry lysine sites identified in argininosuccinate synthase (ASS1), a key enzyme in the urea cycle, are regulated by SIRT5. Mutation of the high-stoichiometry lysine in ASS1 to succinyl-mimetic glutamic acid significantly decreased its enzymatic activity. Metabolomics profiling confirms that SIRT5 deficiency decreases urea cycle activity in liver. Importantly, SIRT5 deficiency compromises ammonia tolerance, which can be reversed by the overexpression of wild-type, but not succinyl-mimetic, ASS1. Therefore, lysine succinylation is functionally important in ammonia metabolism.
proteins
2025-09-20 | Functional Profiling of 2,193 ASS1 Missense Variants: Insights into Variant Pathogenicity and Epistatic Interactions in Citrullinemia Type I
Sequence variants in the urea cycle gene argininosuccinate synthase (ASS1) cause Citrullinemia type 1 (CTLN1), a rare autosomal recessive disease. Mechanistically, reduction in argininosuccinate synthetase (ASS) enzyme activity impairs the urea cycle, leading to an accumulation of citrulline and neurotoxic ammonia. Disease severity varies according to the degree of enzyme impairment, ranging from severe neonatal forms (classic citrullinemia) to milder, late-onset forms that may manifest in childhood or adulthood. We established a high-throughput yeast functional assay of human ASS and individually measured the impact of 2193 amino acid substitutions, representing 90% of all single nucleotide variant (SNV)-accessible substitutions. When benchmarked against existing clinical variant annotation, our assay distinguishes known benign variants from strong loss of function pathogenic variants, enabling identification of a functional score threshold below which variants show clinically relevant impairment of ASS activity. Using the ACMG OddsPath framework, our assay meets PS3_supporting criteria for pathogenicity classification and achieves full PS3-level strength when variants observed as homozygotes in other primates are used as benign proxies for calibration. These results provide direct functional evidence to inform reclassification of ASS1 missense variants, supporting their clinical interpretation and diagnostic utility. Mapping functional scores onto the protein structure, we confirmed that residues involved in catalysis are highly sensitive to substitution. In addition, we identified residues from adjacent subunits of the ASS homotetramer that form compound active sites. Assaying these positions revealed a capacity for intragenic complementation consistent with a variant sequestration model: a form of positive epistasis in which deleterious variants from different subunits are sequestered into only a subset of active sites, restoring function in the remaining variant-free sites. The discovery of intragenic complementation in ASS reveals a novel mode of functional interaction with clinical implications for interpreting variant combinations in heterozygous individuals.
2022-03-15 | Phase 1 trial of ADI-PEG 20 and liposomal doxorubicin in patients with metastatic solid tumors.
Arginine depletion interferes with pyrimidine metabolism and DNA damage repair pathways. Preclinical data demonstrated that depletion of arginine by PEGylated arginine deiminase (ADI-PEG 20) enhanced liposomal doxorubicin (PLD) cytotoxicity in cancer cells with argininosuccinate synthase 1 (ASS1) deficiency. The objective of this study was to assess safety and tolerability of ADI-PEG 20 and PLD in patients with metastatic solid tumors. Patients with advanced ASS1-deficient solid tumors were enrolled in this phase 1 trial of ADI-PEG 20 and PLD following a 3 + 3 design. Eligible patients were given intravenous PLD biweekly and intramuscular (IM) ADI-PEG 20 weekly. Toxicity and efficacy were evaluated according to the Common Terminology Criteria for Adverse Events (version 4.0) and Response Evaluation Criteria in Solid Tumors (version 1.1), respectively. Of 15 enrolled patients, 9 had metastatic HER2-negative breast carcinoma. We observed no dose-limiting toxicities or treatment-related deaths. One patient safely received 880 mg/m2 PLD in this study and 240 mg/m2 doxorubicin previously. Treatment led to stable disease in 9 patients and was associated with a median progression-free survival time of 3.95 months in 15 patients. Throughout the duration of treatment, decreased arginine and increased citrulline levels in peripheral blood remained significant in a majority of patients. We detected no induction of anti-ADI-PEG 20 antibodies by week 8 in one third of patients. Concurrent IM injection of ADI-PEG 20 at 36 mg/m2 weekly and intravenous infusion of PLD at 20 mg/m2 biweekly had an acceptable safety profile in patients with advanced ASS1-deficient solid tumors. Further evaluation of this combination is under discussion.
2021-03-05 | Phase 1 trial of ADI-PEG20 plus cisplatin in patients with pretreated metastatic melanoma or other advanced solid malignancies
Abstract Background Arginine depletion interferes with pyrimidine metabolism and DNA damage-repair pathways, and pairing arginine deiminase pegylated with 20,000-molecular-weight polyethylene glycol (ADI-PEG20) with platinum enhances cytotoxicity in vitro and in vivo in arginine auxotrophs. Methods This single-centre, Phase 1 trial was conducted using a 3 + 3 dose escalation designed to assess safety, tolerability and determine the recommended Phase 2 dose (RP2D) of ADI-PEG20. Results We enrolled 99 patients with metastatic argininosuccinate synthetase 1 (ASS1) deficient malignancies. We observed no dose-limiting toxic effects or treatment-related mortality. Three percent of patients discontinued treatment because of toxicity. After treatment, 5% (5/99) of patients had partial responses, and 41% had stable disease. The median progression-free and overall survival durations were 3.62 and 8.06 months, respectively. Substantial arginine depletion and citrulline escalation persisted in most patients through weeks 24 and 8, respectively. Tumour responses were associated with anti-ADI-PEG20 antibody levels at weeks 8 and 16 ( p = 0.031 and p = 0.0357, respectively). Conclusion Concurrently administered ADI-PEG20 and cisplatin had an acceptable safety profile and had shown antitumour activity against metastatic ASS1-deficient solid tumours. Further evaluation of this treatment combination is warranted.
2020-06-19 | A Phase I Study of Pegylated Arginine Deiminase (Pegargiminase), Cisplatin, and Pemetrexed in Argininosuccinate Synthetase 1-Deficient Recurrent High-grade Glioma.
Patients with recurrent high-grade gliomas (HGG) are usually managed with alkylating chemotherapy ± bevacizumab. However, prognosis remains very poor. Preclinically, we showed that HGGs are a target for arginine depletion with pegargiminase (ADI-PEG20) due to epimutations of argininosuccinate synthetase (ASS1) and/or argininosuccinate lyase (ASL). Moreover, ADI-PEG20 disrupts pyrimidine pools in ASS1-deficient HGGs, thereby impacting sensitivity to the antifolate, pemetrexed. We expanded a phase I trial of ADI-PEG20 with pemetrexed and cisplatin (ADIPEMCIS) to patients with ASS1-deficient recurrent HGGs (NCT02029690). Patients were enrolled (01/16-06/17) to receive weekly ADI-PEG20 36 mg/m2 intramuscularly plus pemetrexed 500 mg/m2 and cisplatin 75 mg/m2 intravenously once every 3 weeks for up to 6 cycles. Patients with disease control were allowed ADI-PEG20 maintenance. The primary endpoints were safety, tolerability, and preliminary estimates of efficacy. Ten ASS1-deficient heavily pretreated patients were treated with ADIPEMCIS therapy. Treatment was well tolerated with the majority of adverse events being Common Terminology Criteria for Adverse Events v4.03 grade 1-2. The best overall response was stable disease in 8 patients (80%). Plasma arginine was suppressed significantly below baseline with a reciprocal increase in citrulline during the sampling period. The anti-ADI-PEG20 antibody titer rose during the first 4 weeks of treatment before reaching a plateau. Median progression-free survival (PFS) was 5.2 months (95% confidence interval (CI), 2.5-20.8) and overall survival was 6.3 months (95% CI, 1.8-9.7). In this recurrent HGG study, ADIPEMCIS was well tolerated and compares favorably to historical controls. Additional trials of ADI-PEG20 in HGG are planned.
2015-08-26 | Argininosuccinate synthetase (ASS) deficiency in high-grade pulmonary neuroendocrine carcinoma: an opportunity for personalized targeted therapy.
Cells deficient in argininosuccinate synthetase (ASS) must absorb the arginine they need for growth from circulating blood. Treatment with pegylated arginine deiminase (ADI-PEG 20) selectively eliminates arginine from the circulation and has shown some efficacy against ASS-deficient tumors including small cell lung cancer (SCLC). We sought to assess ASS expression in a cohort of high-grade pulmonary neuroendocrine carcinomas (PNEC) which include SCLC and large cell neuroendocrine carcinoma (LCNEC). Sixty-nine PNEC (49 SCLC and 20 LCNEC) were retrieved from our pathology archives. Formalin-fixed paraffin-embedded sections of the 54 primary tumors, 15 metastases and appropriate positive and negative controls were immunostained using an ASS-specific monoclonal antibody. Positive staining in <30 % of the tumor was scored as weak; staining in ≥30 % of the tumor was scored as strong. The absence of staining in the tumor was recorded as ASS negative. 58 % of the PNEC including 61.2 % of the SCLC and 50 % of the LCNEC were ASS negative. These ASS-negative tumors included 63 % of the primary and 40 % of the metastatic lesions tested. More than 50 % of the high-grade PNEC tested lack immunohistochemically detectable ASS, suggesting that they are auxotrophic for arginine and potential candidates for arginine deprivation therapy. PNEC comprise about 25 % of primary lung cancers and have a 5-year overall survival of only 5-10 %, underscoring the need for new and more effective therapies. Immunostaining for ASS has potential to improve the selection of patients with PNEC for arginine deprivation therapy with ADI-PEG 20.
oligonucleotides
2026-05-13 | RNA-LNP-mediated in vivo prime editing corrects disease phenotypes in a mouse model of citrullinemia type I.
Citrullinemia type I (CTLN1) is a severe urea cycle disorder caused by pathogenic variants in the ASS1 (argininosuccinate synthetase 1) gene, for which liver transplantation remains the only curative option. Here, we used prime editing to correct the Ass1fold mouse model of CTLN1. Adeno-associated virus (AAV)-mediated delivery of the PE7 prime editor with an optimized prime editing guide RNA (pegRNA) achieved 71 and 54% correction of the pathogenic Ass1 mutation in hepatocytes of neonates and juveniles, respectively. Delivery of mRNA-encoded PE7 and synthetic pegRNA via lipid nanoparticles (LNPs) resulted in 24% correction after a single 3 mg kg-1 dose in neonates and 13% after three 4 mg kg-1 doses in juveniles. All treated groups showed full normalization of survival and of blood citrulline and ammonia concentrations, with restored urea cycle function and correction of natural behavior defects. Consistent with these findings, immunostaining demonstrated restoration of wild-type-like ASS1 protein localization in functionally relevant periportal and intermediate-zone hepatocytes. Editing was confined to the liver, with minimal indel formation and off-target activity and only transient elevations in liver enzymes. In a cellular reporter system, 6 of 15 recurrent human pathogenic ASS1 mutations studied, including the most common ASS1G390R variant, were corrected with similar or higher efficiencies than Ass1fold. These findings highlight prime editing as a precise and potentially curative treatment strategy for individuals with CTLN1 and other genetic liver diseases.
2024-10-25 | Exploring RNA therapeutics for urea cycle disorders.
RNA has triggered a significant shift in modern medicine, providing a promising way to revolutionize disease treatment methods. Different therapeutic RNA modalities have shown promise to replace, supplement, correct, suppress, or eliminate the expression of a targeted gene. Currently, there are 22 RNA-based drugs approved for clinical use, including the COVID-19 mRNA vaccines, whose unprecedented worldwide success has meant a definitive boost in the RNA research field. Urea cycle disorders (UCD), liver diseases with high mortality and morbidity, may benefit from the progress achieved, as different genetic payloads have been successfully targeted to liver using viral vectors, N-acetylgalactosamine (GalNAc) conjugations or lipid nanoparticles (LNP). This review explores the potential of RNA-based medicines for UCD and the ongoing development of applications targeting specific gene defects, enzymes, or transporters taking part in the urea cycle. Notably, LNP-formulated mRNA therapy has been assayed preclinically for citrullinemia type I (CTLN1), adolescent and adult citrin deficiency, argininosuccinic aciduria, arginase deficiency and ornithine transcarbamylase deficiency, in the latter case has progressed to the clinical trials phase.
2024-01-22 | ASS1 deficiency is associated with impaired neuronal differentiation in zebrafish larvae.
Citrullinemia type 1 (CTLN1) is a rare autosomal recessive urea cycle disorder caused by deficiency of the cytosolic enzyme argininosuccinate synthetase 1 (ASS1) due to pathogenic variants in the ASS1 gene located on chromosome 9q34.11. Even though hyperammenomia is considered the major pathomechanistic factor for neurological impairment and cognitive dysfunction, a relevant subset of individuals presents with a neurodegenerative course in the absence of hyperammonemic decompensations. Here we show, that ASS1 deficiency induced by antisense-mediated knockdown of the zebrafish ASS1 homologue is associated with defective neuronal differentiation ultimately causing neuronal cell loss and consecutively decreased brain size in zebrafish larvae in vivo. Whereas ASS1-deficient zebrafish larvae are characterized by markedly elevated concentrations of citrulline - the biochemical hallmark of CTLN1, accumulation of L-citrulline, hyperammonemia or therewith associated secondary metabolic alterations did not account for the observed phenotype. Intriguingly, coinjection of the human ASS1 mRNA not only normalized citrulline concentration but also reversed the morphological cerebral phenotype and restored brain size, confirming conserved functional properties of ASS1 across species. The results of the present study imply a novel, potentially non-enzymatic (moonlighting) function of the ASS1 protein in neurodevelopment.
2024-01-10 | mRNA therapy corrects defective glutathione metabolism and restores ureagenesis in preclinical argininosuccinic aciduria
The urea cycle enzyme argininosuccinate lyase (ASL) enables the clearance of neurotoxic ammonia and the biosynthesis of arginine. Patients with ASL deficiency present with argininosuccinic aciduria, an inherited metabolic disease with hyperammonemia and a systemic phenotype coinciding with neurocognitive impairment and chronic liver disease. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics and in vivo positron emission tomography (PET) imaging using ( S )-4-(3- 18 F-fluoropropyl)- l -glutamate ([ 18 F]FSPG). Up-regulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways. To assess hepatic glutathione dysregulation and liver disease, we present [ 18 F]FSPG PET as a noninvasive diagnostic tool to monitor therapeutic response in argininosuccinic aciduria. Human hASL mRNA encapsulated in lipid nanoparticles improved glutathione metabolism and chronic liver disease. In addition, hASL mRNA therapy corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis. These findings provide mechanistic insights in liver glutathione metabolism and support clinical translation of mRNA therapy for argininosuccinic aciduria.
2023-12-14 | Ex vivo precision-cut liver slices model disease phenotype and monitor therapeutic response for liver monogenic diseases
Background In academic research and the pharmaceutical industry, in vitro cell lines and in vivo animal models are considered as gold standards in modelling diseases and assessing therapeutic efficacy. However, both models have intrinsic limitations, whilst the use of precision-cut tissue slices can bridge the gap between these mainstream models. Precision-cut tissue slices combine the advantage of high reproducibility, studying all cell sub-types whilst preserving the tissue matrix and extracellular architecture, thereby closely mimicking a mini-organ. This approach can be used to replicate the biological phenotype of liver monogenic diseases using mouse models. Methods Here, we describe an optimised and easy-to-implement protocol for the culture of sections from mouse livers, enabling its use as a reliable ex-vivo model to assess the therapeutic screening of inherited metabolic diseases Results We show that precision-cut liver sections can be a reliable model for recapitulating the biological phenotype of inherited metabolic diseases, exemplified by common urea cycle defects such as citrullinemia type 1 and argininosuccinic aciduria, caused by argininosuccinic synthase (ASS1) and argininosuccinic lyase (ASL) deficiencies respectively. Conclusions Therapeutic response to gene therapy such as messenger RNA replacement delivered via lipid nanoparticles can be monitored, demonstrating that precision-cut liver sections can be used as a preclinical screening tool to assess therapeutic response and toxicity in monogenic liver diseases.
small molecules
2026-01-01 | P047: Incidental identification of citrullinemia type 1 through carrier screening post liver transplant
Outcome and Follow-Up: Following the addition of carglumic acid, we were able to gradually wean our patient off all additional urea cycle disorder (UCD) management -ammonia scavengers and medical formula -over the course of five months.The patient has not experienced any further hyperammonemic episodes.Citrulline supplementation was discontinued at 11 months and plasma citrulline levels have remained normal to date.Glutamine and alanine levels have remained in normal range, with a maximum glutamine level of 671mol/L.At 21 months of age, the patient remains metabolically stable on carglumic acid 53.6mg/kg/day with dietary protein allotment not exceeding 2.2g/kg/day from intact protein without UCD formula.Developmentally he demonstrates mild-to-moderate delays; likely reflecting his hyperammonemia at presentation.Discussion: This case highlights the importance of early recognition and prompt initiation of carglumic acid in this diagnosis.The identified NAGS c.1289T>C variant adds to the catalog of pathogenic variants identified in this gene.Carglumic acid, an analog of N-acetylglutamate, has proven an effective sole treatment for this enzyme deficiency in our patient with sustained metabolic control.Conclusion: Early diagnosis and prompt initiation of carglumic acid therapy are lifesaving and life-altering in NAGS deficiency.This case contributes to the very limited clinical literature about this diagnosis and its long-term management, while adding to the clinical experience a novel variant.
2025-02-03 | BCL-XL Protects ASS1-Deficient Cancers from Arginine Starvation-Induced Apoptosis.
Argininosuccinate synthetase 1 (ASS1) silencing in carcinomas and sarcomas leads to a dependence on extracellular arginine for survival. Arginine deprivation therapies, such as PEGylated arginine deiminase (ADI-PEG20), have shown limited effectiveness, which may be due to underlying mechanisms that inhibit apoptosis. The effects of ADI-PEG20 on cell-cycle regulation, apoptosis, and BCL-XL-mediated survival pathways in ASS1-deficient cancer cells were determined. The mechanism of cell death protection was determined by assessing caspase and PARP cleavage, CDK2 activity, MCL1 expression, and the interactions among BCL-XL, BAX, and BAK. In vitro synergy was determined, and in vivo efficacy was modeled. Treatment with ADI-PEG20 led to reduced CDK2 activity and inhibited cell-cycle progression but did not induce significant cell death. BCL-XL was found to bind to BAX and BAK, preventing the initiation of apoptosis despite arginine starvation. Inhibition of BCL-XL allowed proapoptotic BAX and BAK to initiate the intrinsic apoptosis pathway, leading to increased cell death. This was found to be synergistic in vitro and efficacious in combination in vivo. The study identifies BCL-XL as a key factor limiting the efficacy of arginine starvation therapies. Combining BCL-XL inhibitors with arginine deprivation strategies may overcome this resistance and enhance therapeutic outcomes. These findings provide a strong preclinical rationale for testing this combination approach in phase 1 clinical trials for ASS1-deficient cancers.
2024-09-16 | Data from Discovery and targeting of a noncanonical mechanism of sarcoma resistance to ADI-PEG20 mediated by the microenvironment
<div>Abstract<p>Purpose: Many cancers lack argininosuccinate synthetase 1 (ASS1), the rate limiting enzyme of arginine biosynthesis. This deficiency causes arginine auxotrophy, targetable by extracellular arginine-degrading enzymes such as ADI-PEG20. Long-term tumor resistance has thus far been attributed solely to ASS1 re-expression. This study examines the role of ASS1 silencing on tumor growth and initiation and identifies a noncanonical mechanism of resistance, aiming to improve clinical responses to ADI-PEG20. Experimental Design: Tumor initiation and growth rates were measured for a spontaneous <i>Ass1</i> knockout (KO) murine sarcoma model. Tumor cell lines were generated, and resistance to arginine deprivation therapy was studied <i>in vitro </i>and<i> in vivo</i>. Results: Conditional <i>Ass1</i> KO affected neither tumor initiation nor growth rates in a sarcoma model, contradicting the prevalent idea that ASS1 silencing confers a proliferative advantage. <i>Ass1</i> KO cells grew robustly through arginine starvation <i>in vivo, </i>while ADI-PEG20 remained completely lethal <i>in vitro</i>, evidence that pointed toward a novel mechanism of resistance mediated by the microenvironment. Co-culture with <i>Ass1</i>-competent fibroblasts rescued growth through macropinocytosis of vesicles and/or cell fragments, followed by recycling of protein-bound arginine through autophagy/lysosomal degradation. Inhibition of either macropinocytosis or autophagy/lysosomal degradation abrogated this growth support effect <i>in vitro</i> and <i>in vivo</i>. Conclusions: Noncanonical, ASS1-independent tumor resistance to ADI-PEG20 is driven by the microenvironment. This mechanism can be targeted by either the macropinocytosis inhibitor imipramine or the autophagy inhibitor chloroquine. These safe, widely available drugs should be added to current clinical trials to overcome microenvironmental arginine support of tumors and improve patient outcomes.</p></div>
2023-11-09 | Case report: Diagnosis of ADCY5-related dyskinesia explaining the entire phenotype in a patient with atypical citrullinemia type I
In this case study, we report the case of a 13-year-old girl with citrullinemia type 1 (MIM #215700), an autosomal recessive inherited disorder of the urea cycle, which was confirmed by the identification of a homozygous pathogenic variant in the argininosuccinate synthetase 1 (ASS1) gene. However, the patient presented abnormal hyperkinetic movements with global developmental delay and clinical signs that were not fully consistent with those of citrullinemia type 1 or with those of her siblings with isolated citrullinemia type 1. Exome sequencing showed the presence of a de novo heterozygous pathogenic variant in the adenylate cyclase type 5 (ADCY5) gene. The variant confirmed the overlap with the so-called ADCY5-related dyskinesia with orofacial involvement, which is autosomal dominant (MIM #606703), a disorder disrupting the enzymatic conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). In addition to the citrullinemia-related low-protein diet and arginine supplementation, the identification of this second disease led to the introduction of a treatment with caffeine, which considerably improved the dyskinesia neurological picture. In conclusion, this case highlights the importance of clinical-biological confrontation for the interpretation of genetic variants, as one hereditary metabolic disease may hide another with therapeutic consequences.This article reports the misleading superposition of two inherited metabolic diseases, showing the importance of clinical-biological confrontation in the interpretation of genetic variants.
2023-09-01 | Evaluation of the Neurodevelopmental Status for Urea Cycle Disorders: Based on Clinical Experience
Aim: Urea cycle disorders (UCD) still have poor neurological outcomes despite early diagnosis and treatment.We aimed to present the neurological outcomes of UCD patients and to determine the main simple and accessible factors affecting these outcomes. Materials and Methods:This was a descriptive cross-sectional study conducted in two pediatric metabolism centers on 29 patients from 25 unrelated families who were diagnosed and followed with UCD based on clinical presentation, neurological parameters, biochemical measurements, and molecular analysis.Results: Within the study population, the most common diagnosis was argininosuccinate synthase deficiency in 13 (44.82%)patients, followed by N-acetylglutamate synthase deficiency in five patients (17.24%), ornithine transcarbamylase deficiency in four patients (13.79%), arginase 1 deficiency in three patients (10.34%), carbamoyl phosphate synthase 1 deficiency in three patients (10.34%), and argininosuccinate lyase deficiency in one patient (3.44%).Peak ammonia levels were observed to be significantly higher in those patients with delayed milestones and patients who had Denver II <-2 standard deviation score results (p=0.032,p=0.026).Effect sizes were large in both groups.Delayed milestones were noted in 17 (94.4%) of the cases with peak ammonia >500 μmol/L (n=18).Those patients with abnormal neurological parameters had a significantly higher mean number of hyperammonemic episodes per year.Extracorporeal detoxification was given to eight patients, in combination with therapeutic hypothermia in two patients.Rapid regression was observed in brain edema in those who underwent therapeutic hypothermia. Conclusion:Our study emphasizes the effect of peak ammonia levels and the frequency of hyperammonemic episodes on neurological outcomes.There were still poor neurocognitive outcomes despite extracorporeal detoxification.This highlights the need to reassess current treatment strategies, including the threshold for starting extracorporeal detoxification if ammonia levels exceed 500 μmol/L.The use of therapeutic hypothermia by experienced teams may be promising due to its brain edema-reducing effects.
other
2022-11-16 | Managing recurrent portal steal in auxiliary liver transplantation for non-cirrhotic metabolic liver disease.
APOLT has been proposed as a treatment modality for certain types of NCMLD. While the short-term outcomes of this operation have been comparable with orthotopic LT, its long-term outcomes have sparsely been reported. We present one such case of Citrullinemia type I who underwent APOLT and developed recurrent PS. A 2-year-old male child with a diagnosis of Citrullinemia type I underwent APOLT with a left lateral segment from a split deceased donor liver, and his postoperative period was unremarkable. Ammonia-lowering agents were stopped 1 week following the operation and the child was discharged home on a normal diet. Four years following APOLT, the child presented with altered sensorium and seizures. A diagnosis of PS was made. Subsequent to an embolization of the native liver's right anterior portal vein his sensorium improved and he remained clinically stable on a normal diet. Six years following the APOLT, the child again presented with features of acute encephalopathy. Imaging was suggestive of PS. A portal vein embolization of the native portal vein was performed and the child's clinical condition improved. At 6 months' follow-up, the child remains well on a normal diet. While the early impediments in this technique may have been overcome, in the absence of any realistic clinical application gene therapy, the debate of long-term phenotypic metabolic correction for NCMLD by APOLT needs to be revisited.
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Drug Discovery Landscape
4 orphan drug designations for Citrullinemia type I, including 1 approved therapy.
4 orphan drug designations for Citrullinemia type I, including 1 approved therapy.
Drug | Therapy type | Regulator | Orphan designation | Approval | Sponsor |
|---|---|---|---|---|---|
Sodium benzoate, sodium phenylacetate | small molecules | EMA | 2019-05-29 | — | Dipharma B.V. |
Sodium phenylbutyrate [Pheburane] | small molecules | EMA | 2012-02-09 | — | Lucane Pharma |
Human heterologous liver cells (for infusion) | cell therapies | EMA | 2010-12-17 | — | Promethera Biosciences |
Glyceryl tri-(4-phenylbutyrate) [Ravicti] | small molecules | EMA | 2010-06-10 | 2015-12-01 | Immedica Pharma AB |
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