RARE DISEASE
Citrullinemia
Citrullinemia
Citrullinemia
Drug discovery
2
drugs
With orphan designations
Overview
Citrullinemia is an autosomal recessive urea cycle disorder characterized by toxic ammonia accumulation due to impaired argininosuccinate synthetase (type I) or citrin transporter (type II). Neonatal type I presents with hyperammonemia, lethargy, seizures, and coma, while type II often manifests in adulthood with neuropsychiatric symptoms. Diagnosis involves elevated plasma citrulline and genetic testing. Management includes dietary modifications, ammonia scavengers, and liver transplantation. Long-term complications include intellectual disability and hepatic dysfunction [1][3][11][14].
Burden
Neonatal type I mortality reaches 20% in developed nations despite treatment [4][12].
Chronic neurocognitive deficits, growth impairment, and recurrent hyperammonemic crises necessitate lifelong monitoring [9][11][19].
Liver transplantation remains the only cure but carries surgical risks and requires immunosuppression [3][4].
Therapies
Categories: rare genetic diseases, rare inborn errors of metabolism
Research Papers
225 drug discovery papers about Citrullinemia, with 3 first-in-class and 7 next-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
225 drug discovery papers about Citrullinemia, with 3 first-in-class and 7 next-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
categories:
Small molecules
small molecules
2026-03-19 | Impact of long-term nitrogen scavenger therapy on clinical outcome in individuals with urea cycle disorders.
Principles of long-term medical management in individuals with urea cycle disorders (UCDs) encompass (1) a low protein diet, (2) supplementation of arginine and/or citrulline along with essential amino acids, nutrients, vitamins and trace elements, and (3) use of nitrogen scavenging agents to reduce recurrent hyperammonemic events (HAEs). These principles aim at providing metabolic stability, elimimation of chronic complications, and achievement of normal development as well as growth. A retrospective comparative analysis was performed by studying 138 individuals with male ornithine transcarbamylase deficiency (mOTC-D), citrullinemia type 1 (CTLN1) and argininosuccinic aciduria (ASA) based on in vitro residual enzymatic activity for severity-adjustment. Results show that individuals with mOTC-D, CTLN1 and ASA are at risk of progressive linear growth impairment, recurrent annual HAEs and an unfavorable neurocognitive outcome despite being under long-term nitrogen scavenging pharmacotherapy. No overall superiority among existing nitrogen scavenging agents with regard to the individual's metabolic stability, linear growth impairment and poor neurocognitive outcome was observed. Novel therapeutic strategies are urgently needed to ultimately improve health outcomes in individuals with UCDs in order to sufficiently meet guideline-specific goals.
2026-03-11 | Looking Beyond Severe Hypertriglyceridemia when Diagnosing Adult-Onset Citrullinemia Type II.
Adult-onset citrullinemia type II (CTLN2) is a rare autosomal recessive urea cycle disorder caused by mutations in the solute carrier family 25 member 13 (SLC25A13) gene, which encodes citrin-a mitochondrial transporter involved in the malate-aspartate shuttle. In adults, CTLN2 may present atypically as isolated hypertriglyceridemia, often misattributed to secondary dyslipidaemia. A 30-year-old Vietnamese male with longstanding severe hypertriglyceridemia, first identified at age 13, was referred after an episode of acute pancreatitis at age 28. At presentation, plasma triglyceride levels reached 34.61 mmol/l. Secondary causes were excluded. Genetic testing via next-generation sequencing revealed compound heterozygous SLC25A13 mutations: c.2T>C and c.1638_1660dup, resulting in p.Met1Thr and p.Ala554GlyfsTer17, confirming the diagnosis of CTLN2. Initiation of a low-carbohydrate, high-protein, low-fat diet combined with fenofibrate (145 mg/day) led to a rapid reduction in triglyceride levels, normalizing within two weeks and remaining stable over three months. The treatment was well tolerated, with no reported adverse effects. CTLN2 should be considered in young adults with persistent, unexplained, and severe hypertriglyceridemia. Dietary modification constitutes the cornerstone of management, with fibrates playing a supportive role. Persistent, unexplained, and severe hypertriglyceridemia in young adults should prompt consideration of inherited metabolic disorders, including adult-onset citrullinemia type II (CTLN2).Genetic testing to detect compound heterozygous or homozygous SLC25A13 variants is essential for definitive diagnosis of CTLN2.In CTLN2, dietary management with a low-carbohydrate, high-protein, low-fat represents the cornerstone, with fibrates serving as adjunctive lipid-lowering therapy.
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
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.
gene therapies
2024-01-05 | Treatment and management for children with urea cycle disorder in chronic stage.
Urea cycle disorder (UCD) is a group of inherited metabolic diseases with high disability or fatality rate, which need long-term drug treatment and diet management. Except those with Citrin deficiency or liver transplantation, all pediatric patients require lifelong low protein diet with safe levels of protein intake and adequate energy and lipids supply for their corresponding age; supplementing essential amino acids and protein-free milk are also needed if necessary. The drugs for long-term use include nitrogen scavengers (sodium benzoate, sodium phenylbutyrate, glycerol phenylbutyrate), urea cycle activation/substrate supplementation agents (N-carbamylglutamate, arginine, citrulline), etc. Liver transplantation is recommended for pediatric patients not responding to standard diet and drug treatment, and those with severe progressive liver disease and/or recurrent metabolic decompensations. Gene therapy, stem cell therapy, enzyme therapy and other novel technologies may offer options for treatment in UCD patients. The regular biochemical assessments like blood ammonia, liver function and plasma amino acid profile are needed, and physical growth, intellectual development, nutritional intake should be also evaluated for adjusting treatment in time. 尿素循环障碍（UCD）是一组致死、致残率较高的遗传代谢病，需要长期饮食和药物治疗及管理。除希特林蛋白缺乏症和行肝移植治疗的患儿，其他慢性期患儿均需要终身低蛋白饮食，保证其相应年龄的安全蛋白质摄入量以及充足的碳水和脂肪的供能比，必要时补充必需氨基酸及无蛋白奶粉；药物治疗主要包括氮清除剂（苯甲酸钠、苯丁酸钠、苯丁酸甘油酯）、尿素循环激活/底物补充剂（N-氨基甲酰谷氨酸、精氨酸、瓜氨酸）等。规范饮食及药物治疗后未达预期效果、出现严重进展性肝病或出现反复发作的患儿建议行肝移植。基因疗法、干细胞疗法和酶替代疗法等新技术可能是UCD患儿治疗的新选择。UCD患儿需要定期检测血氨、肝功能和血氨基酸等生化指标，并评估体格生长、智力发育和营养摄入情况，及时调整治疗方案。. Urea cycle disorder (UCD) is a group of inherited metabolic diseases with high disability or fatality rate, which need long-term drug treatment and diet management. Except those with Citrin deficiency or liver transplantation, all pediatric patients require lifelong low protein diet with safe levels of protein intake and adequate energy and lipids supply for their corresponding age; supplementing essential amino acids and protein-free milk are also needed if necessary. The drugs for long-term use include nitrogen scavengers (sodium benzoate, sodium phenylbutyrate, glycerol phenylbutyrate), urea cycle activation/substrate supplementation agents (N-carbamylglutamate, arginine, citrulline), etc. Liver transplantation is recommended for pediatric patients not responding to standard diet and drug treatment, and those with severe progressive liver disease and/or recurrent metabolic decompensations. Gene therapy, stem cell therapy, enzyme therapy and other novel technologies may offer options for treatment in UCD patients. The regular biochemical assessments like blood ammonia, liver function and plasma amino acid profile are needed, and physical growth, intellectual development, nutritional intake should be also evaluated for adjusting treatment in time.
2023-09-25 | Lethality rescue and long-term amelioration of a citrullinemia type I mouse model by neonatal gene-targeting combined to SaCRISPR-Cas9.
Citrullinemia type I is a rare autosomal-recessive disorder caused by deficiency of argininosuccinate synthetase (ASS1). The clinical presentation includes the acute neonatal form, characterized by ammonia and citrulline accumulation in blood, which may lead to encephalopathy, coma, and death, and the milder late-onset form. Current treatments are unsatisfactory, and the only curative treatment is liver transplantation. We permanently modified the hepatocyte genome in lethal citrullinemia mice (Ass1fold/fold) by inserting the ASS1 cDNA into the albumin locus through the delivery of two AAV8 vectors carrying the donor DNA and the CRISPR-Cas9 platform. The neonatal treatment completely rescued mortality ensuring survival up to 5 months of age, with plasma citrulline levels significantly decreased, while plasma ammonia levels remained unchanged. In contrast, neonatal treatment with a liver-directed non-integrative AAV8-AAT-hASS1 vector failed to improve disease parameters. To model late-onset citrullinemia, we dosed postnatal day (P) 30 juvenile animals using the integrative approach, resulting in lifespan improvement and a minor reduction in disease markers. Conversely, treatment with the non-integrative vector completely rescued mortality, reducing plasma ammonia and citrulline to wild-type values. In summary, the integrative approach in neonates is effective, although further improvements are required to fully correct the phenotype. Non-integrative gene therapy application to juvenile mice ensures a stable and very efficient therapeutic effect.
2022-11-29 | Gene Therapy in Combination with Nitrogen Scavenger Pretreatment Corrects Biochemical and Behavioral Abnormalities of Infant Citrullinemia Type 1 Mice
Citrullinemia type I (CTLN1) is a rare autosomal recessive disorder caused by mutations in the gene encoding argininosuccinate synthetase 1 (ASS1) that catalyzes the third step of the urea cycle. CTLN1 patients suffer from impaired elimination of nitrogen, which leads to neurotoxic levels of circulating ammonia and urea cycle byproducts that may cause severe metabolic encephalopathy, death or irreversible brain damage. Standard of care (SOC) of CTLN1 consists of daily nitrogen-scavenger administration, but patients remain at risk of life-threatening decompensations. We evaluated the therapeutic efficacy of a recombinant adeno-associated viral vector carrying the ASS1 gene under the control of a liver-specific promoter (VTX-804). When administered to three-week-old CTLN1 mice, all the animals receiving VTX-804 in combination with SOC gained body weight normally, presented with a normalization of ammonia and reduction of citrulline levels in circulation, and 100% survived for 7 months. Similar to what has been observed in CTLN1 patients, CTLN1 mice showed several behavioral abnormalities such as anxiety, reduced welfare and impairment of innate behavior. Importantly, all clinical alterations were notably improved after treatment with VTX-804. This study demonstrates the potential of VTX-804 gene therapy for future clinical translation to CTLN1 patients.
2019-10-01 | Robust, Long-Term Culture of Endoderm-Derived Hepatic Organoids for Disease Modeling
Organoid technologies have become a powerful emerging tool to model liver diseases, for drug screening, and for personalized treatments. These applications are, however, limited in their capacity to generate functional hepatocytes in a reproducible and efficient manner. Here, we generated and characterized the hepatic organoid (eHEPO) culture system using human induced pluripotent stem cell (iPSC)-derived EpCAM-positive endodermal cells as an intermediate. eHEPOs can be produced within 2 weeks and expanded long term (>16 months) without any loss of differentiation capacity to mature hepatocytes. Starting from patient-specific iPSCs, we modeled citrullinemia type 1, a urea cycle disorder caused by mutations in the argininosuccinate synthetase (ASS1) enzyme. The disease-related ammonia accumulation phenotype in eHEPOs could be reversed by the overexpression of the wild-type ASS1 gene, which also indicated that this model is amenable to genetic manipulation. Thus, eHEPOs are excellent unlimited cell sources to generate functional hepatic organoids in a fast and efficient manner.
2019-06-19 | Insights into Gene Therapy for Urea Cycle Defects by Mathematical Modeling
Metabolic liver diseases are attractive gene therapy targets that necessitate reconstitution of enzymatic activity in functionally complex biochemical pathways. The levels of enzyme activity required in individual hepatocytes and the proportion of the hepatic cell mass that must be gene corrected for therapeutic benefit vary in a disease-dependent manner that is difficult to predict. While empirical evaluation is inevitably required, useful insights can nevertheless be gained from knowledge of disease pathophysiology and theoretical approaches such as mathematical modeling. Urea cycle defects provide an excellent example. Building on a previously described one-compartment model of the urea cycle, we have constructed a two-compartment model that can simulate liver-targeted gene therapy interventions using the computational program Mathematica. The model predicts that therapeutically effective reconstitution of ureagenesis will correlate most strongly with the proportion of the hepatic cell mass transduced rather than the level of enzyme-encoding transgene expression achieved in individual hepatocytes. Importantly, these predictions are supported by experimental data in mice and human genotype/phenotype correlations. The most notable example of the latter is ornithine transcarbamylase deficiency (X-linked) where impairment of ureagenesis in male and female patients is closely simulated by the one- and two-compartment models, respectively. Collectively, these observations support the practical value of mathematical modeling in evaluation of the disease-specific gene transfer challenges posed by complex metabolic phenotypes.
cell therapies
2026-06-04 | Sudden Acute Liver Failure in Citrullinemia Type 1: An Argument for Earlier Liver Transplantation?
Acute liver failure has been described at initial presentation of citrullinemia type 1, but recurrent hepatic decompensations during medical treatment have not been reported. We describe 5 children developing episodes of acute liver failure, including one who required emergency liver transplantation. Our findings expand the citrullinemia type 1 phenotype and support early liver transplant referral in selected cases.
2025-05-12 | Supplementation with Kluyveromyces marxianus probiotic yeasts in sows and their piglets in early post- weaning: effects in biological and productive parameters
Weaning in pig production is a stressful event that impacts intestinal biology and increases the risk of infections, impairing piglet development and leading to economic losses. Antibiotic preventive administration has been used to improve the productive parameters, but nowadays this practice has been banned because of its contribution to antimicrobial resistance generation. So, alternatives are urgently needed. Kluyveromyces marxianus CIDCA 9121 has immunomodulatory and convenient biotechnological properties and was studied for preventing post-weaning stress, for which 8 sows were supplemented with 109 CFU per kg of feed from 20 days before piglets´ birth until weaning. Then, piglets were supplemented with 2x109 CFU/ kg of feed for 20 days more. A control group of 8 sows and their piglets not supplemented with yeast was included. Productive parameters were registered from birth to 60 days of age. Five days post-weaning, 5 animals from each group were euthanized; small intestines and blood were taken for histopathology analysis and amino acid analysis respectively. Intestinal content was sampled to determine the main bacterial groups by qPCR. Piglets born from supplemented sows (n = 132) weighed more than controls (n = 130; p < 0,05). During farrowing, there were no differences among groups but 40 days after weaning the supplemented group weighted 10% more than the control group (p < 0,05). We found an increase in citrullinemia and in the small intestine mucosal thickness in supplemented piglets post-weaning indicating better tolerance to post-weaning stress. Our results suggest that K. marxianus CIDCA 9121 is a good candidate for supplementation of gestating sows and postweaning piglets.
2024-11-19 | Development of optimised human iPSC-derived hepatocytes with improved liver function for in vitro metabolic disease modelling and toxicity studies
Abstract Background & Aims Liver disease is a rising cause of mortality worldwide. Primary human hepatocytes (PHH) and hepatocellular cancer cells are currently used in drug development, however, they come with limitations, including limited supply, rapid loss of function, and tumorigenic origin. In addition, current iPSC differentiation protocols lead to the generation of hepatocyte-like cells with compromised liver-related features. We hypothesised that optimisation of iPSC differentiation protocols can lead to the generation of hepatocyte-like cells with improved metabolic functionality for disease modelling and toxicity screening studies. Methods Healthy human iPSCs were differentiated to hepatocyte-like cells (Opti-HEP) using a novel 3-step differentiation protocol. Hepatocyte functionality was assessed, including liver maturity marker expression, urea synthesis, de novo gluconeogenesis, and CYP450 expression, activity, and induction. Suitability of Opti-HEP to predict drug-induced liver injury (DILI) was evaluated by cell viability assays. CRISPR/Cas9 gene editing was employed to generate in vitro inherited metabolic disease models. Results Opti-HEP expressed similar liver maturity marker levels to those seen in primary human hepatocytes (PHH), in addition to functional urea and gluconeogenesis pathways. CYP450 expression and activity were comparable between Opti-HEP and PHH, with both cell types showing similar levels of CYP3A4 induction upon 1α,25-hydroxy-vitamin D3 treatment. Opti-HEP accurately predicted DILI, following treatment with 7 drugs of known DILI liability. CRISPR-derived Opti-HEP harbouring mutations for inherited metabolic disorders (Ornithine Transcarbamylase Deficiency, Progressive Familial Intrahepatic Cholestasis Type 2, Citrullinemia Type 1) recapitulated key pathophysiological disease features, including reduced protein expression, impaired urea secretion, and bile acid transport. Conclusions We demonstrate the generation of optimised iPSC-derived hepatocytes with enhanced liver functionality that is comparable to PHH. These data alongside the expansion capacity and amenability of these cells highlight the opportunities this model can offer in the space of disease modelling and large-scale drug efficacy and hepatotoxicity screening.
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.
2022-09-26 | Modelling urea cycle disorders using iPSCs
Abstract The urea cycle is a liver-based pathway enabling disposal of nitrogen waste. Urea cycle disorders (UCDs) are inherited metabolic diseases caused by deficiency of enzymes or transporters involved in the urea cycle and have a prevalence of 1:35,000 live births. Patients present recurrent acute hyperammonaemia, which causes high rate of death and neurological sequelae. Long-term therapy relies on a protein-restricted diet and ammonia scavenger drugs. Currently, liver transplantation is the only cure. Hence, high unmet needs require the identification of effective methods to model these diseases to generate innovative therapeutics. Advances in both induced pluripotent stem cells (iPSCs) and genome editing technologies have provided an invaluable opportunity to model patient-specific phenotypes in vitro by creating patients’ avatar models, to investigate the pathophysiology, uncover novel therapeutic targets and provide a platform for drug discovery. This review summarises the progress made thus far in generating 2- and 3-dimensional iPSCs models for UCDs, the challenges encountered and how iPSCs offer future avenues for innovation in developing the next-generation of therapies for UCDs.
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.
2025-02-21 | Developing splice-switching oligonucleotides for urea cycle disorder using an integrated diagnostic and therapeutic platform.
Citrin deficiency (CD) is an autosomal recessive urea cycle disorder caused by biallelic loss-of-function variants in the SLC25A13 gene, leading to life-threatening hyperammonemia and hypoglycemia. Variants in deep introns can cause genetic diseases by altering splicing and are often missed by current diagnostic tools. Splice-switching oligonucleotides (SSOs) can resolve certain intronic variants, but patients harboring such variants need to be identified. We present a lean workflow from molecular diagnostics to SSO development to resolve splice-altering variants in deep introns that is applicable to other genetic disorders. A deep intronic-gene panel was designed to identify deep intronic variants. SSOs were then developed and validated in vitro using a minigene assay and induced hepatocytes, and target engagement was verified in vivo by hydrodynamic tail vein injection of minigenes and SSOs. With the deep intronic-gene panel and RNA analysis, we identified a novel SLC25A13 c.469-2922G>T variant that promotes the inclusion of a premature stop codon-containing pseudo-exon, SLC25A13-PE5, thereby causing CD. Using a stepwise rational SSO design approach, we identified potent candidates inhibiting SLC25A13-PE5 at EC50 <2 nM in vitro. Upon conjugating the SSOs with GalNAc (N-acetylgalactosamine), they were validated to rescue normal protein expression and restore ureagenesis and ammonia clearance, key urea cycle functions, in patient-derived induced hepatocytes. In vivo on-target efficacy of the clinical GalNAc-SSO candidate, in the absence of acute toxicity and inflammation, was observed in a mouse model with exogenous hepatic minigene expression. Our data validates a platform to redefine the molecular diagnosis of urea cycle disorders and provides proof-of-concept for a precision therapy for patients with CD, for whom the only effective treatment is liver transplantation. Deep intronic variants are common causes of genetic diseases that are commonly neglected. In this study, we demonstrate an integrated precision diagnostic and therapeutic approach for urea cycle disorders. Specifically, we focus on citrin deficiency, going from the discovery of a novel splice variant in the SLC25A13 gene with our novel deep intronic-gene panel for urea cycle disorders, to the development and in vivo validation of an efficacious splice-switching oligonucleotide candidate for the pathogenic splice variant. We envision the possibility of extrapolating this pipeline to the diagnosis and development of treatments for other rare genetic 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.
2024-07-18 | The therapeutic landscape of citrin deficiency.
Citrin deficiency (CD) is a recessive, liver disease caused by sequence variants in the SLC25A13 gene encoding a mitochondrial aspartate-glutamate transporter. CD manifests as different age-dependent phenotypes and affects crucial hepatic metabolic pathways including malate-aspartate-shuttle, glycolysis, gluconeogenesis, de novo lipogenesis and the tricarboxylic acid and urea cycles. Although the exact pathophysiology of CD remains unclear, impaired use of glucose and fatty acids as energy sources due to NADH shuttle defects and PPARα downregulation, respectively, indicates evident energy deficit in CD hepatocytes. The present review summarizes current trends on available and potential treatments for CD. Baseline recommendation for CD patients is dietary management, often already present as a self-selected food preference, that includes protein and fat-rich food, and avoidance of excess carbohydrates. At present, liver transplantation remains the sole curative option for severe CD cases. Our extensive literature review indicated medium-chain triglycerides (MCT) as the most widely used CD treatment in all age groups. MCT can effectively improve symptoms across disease phenotypes by rapidly supplying energy to the liver, restoring redox balance and inducing lipogenesis. In contrast, sodium pyruvate restored glycolysis and displayed initial preclinical promise, with however limited efficacy in adult CD patients. Ursodeoxycholic acid, nitrogen scavengers and L-arginine treatments effectively address specific pathophysiological aspects such as cholestasis and hyperammonemia and are commonly administered in combination with other drugs. Finally, future possibilities including restoring redox balance, amino acid supplementation, enhancing bioenergetics, improving ureagenesis and mRNA/DNA-based gene therapy are also discussed.
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.
other
2026-06-17 |
Twist variant codon usage for yeast.
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 2,193 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. Under the current ACMG guidelines, inclusion of our data yielded definitive classifications (pathogenic or likely pathogenic) for all 25 ClinVar VUS falling in the functionally impaired range of our assay. 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.
2023-03-16 | Exogenous aralar/slc25a12 can replace citrin/slc25a13 as malate aspartate shuttle component in liver
The deficiency of CITRIN, the liver mitochondrial aspartate–glutamate carrier (AGC), is the cause of four human clinical phenotypes, neonatal intrahepatic cholestasis caused by CITRIN deficiency (NICCD), silent period, failure to thrive and dyslipidemia caused by CITRIN deficiency (FTTDCD), and citrullinemia type II (CTLN2). Clinical symptoms can be traced back to disruption of the malate-aspartate shuttle due to the lack of citrin. A potential therapy for this condition is the expression of aralar, the AGC present in brain, to replace citrin. To explore this possibility we have first verified that the NADH/NAD+ ratio increases in hepatocytes from citrin(−/−) mice, and then found that exogenous aralar expression reversed the increase in NADH/NAD+ observed in these cells. Liver mitochondria from citrin (−/−) mice expressing liver specific transgenic aralar had a small (~ 4–6 nmoles x mg prot−1 x min−1) but consistent increase in malate aspartate shuttle (MAS) activity over that of citrin(−/−) mice. These results support the functional replacement between AGCs in the liver. To explore the significance of AGC replacement in human therapy we studied the relative levels of citrin and aralar in mouse and human liver through absolute quantification proteomics. We report that mouse liver has relatively high aralar levels (citrin/aralar molar ratio of 7.8), whereas human liver is virtually devoid of aralar (CITRIN/ARALAR ratio of 397). This large difference in endogenous aralar levels partly explains the high residual MAS activity in liver of citrin(−/−) mice and why they fail to recapitulate the human disease, but supports the benefit of increasing aralar expression to improve the redox balance capacity of human liver, as an effective therapy for CITRIN deficiency.
2012-01-20 | Substrate Specificity of the Two Mitochondrial Ornithine Carriers Can Be Swapped by Single Mutation in Substrate Binding Site
Mitochondrial carriers are a large family of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has indicated that these transporters have substrate binding sites in a similar location of the central cavity consisting of three major contact points. Here we have characterized mutations of the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 by carrying out transport assays with a set of different substrates. The different substrate specificities of the two isoforms, which share 87% identical amino acids, were essentially swapped by exchanging a single residue located at position 179 that is arginine in ORC1 and glutamine in ORC2. Altogether the substrate specificity changes demonstrate that Arg-179 and Glu-180 of contact point II bind the Cα carboxylate and amino group of the substrates, respectively. Residue Glu-77 of contact point I most likely interacts with the terminal amino group of the substrate side chain. Furthermore, it is likely that all three contact points are involved in the substrate-induced conformational changes required for substrate translocation because Arg-179 is probably connected with Arg-275 of contact point III through Trp-224 by cation-π interactions. Mutations at position 179 also affected the turnover number of the ornithine carrier severely, implying that substrate binding to residue 179 is a rate-limiting step of the catalytic transport cycle. Given that Arg-179 is located in the vicinity of the matrix gate, it is concluded that it is a key residue in the opening of the carrier to the matrix side. Mitochondrial carriers are a large family of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has indicated that these transporters have substrate binding sites in a similar location of the central cavity consisting of three major contact points. Here we have characterized mutations of the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 by carrying out transport assays with a set of different substrates. The different substrate specificities of the two isoforms, which share 87% identical amino acids, were essentially swapped by exchanging a single residue located at position 179 that is arginine in ORC1 and glutamine in ORC2. Altogether the substrate specificity changes demonstrate that Arg-179 and Glu-180 of contact point II bind the Cα carboxylate and amino group of the substrates, respectively. Residue Glu-77 of contact point I most likely interacts with the terminal amino group of the substrate side chain. Furthermore, it is likely that all three contact points are involved in the substrate-induced conformational changes required for substrate translocation because Arg-179 is probably connected with Arg-275 of contact point III through Trp-224 by cation-π interactions. Mutations at position 179 also affected the turnover number of the ornithine carrier severely, implying that substrate binding to residue 179 is a rate-limiting step of the catalytic transport cycle. Given that Arg-179 is located in the vicinity of the matrix gate, it is concluded that it is a key residue in the opening of the carrier to the matrix side.
small molecules
2026-03-19 | Impact of long-term nitrogen scavenger therapy on clinical outcome in individuals with urea cycle disorders.
Principles of long-term medical management in individuals with urea cycle disorders (UCDs) encompass (1) a low protein diet, (2) supplementation of arginine and/or citrulline along with essential amino acids, nutrients, vitamins and trace elements, and (3) use of nitrogen scavenging agents to reduce recurrent hyperammonemic events (HAEs). These principles aim at providing metabolic stability, elimimation of chronic complications, and achievement of normal development as well as growth. A retrospective comparative analysis was performed by studying 138 individuals with male ornithine transcarbamylase deficiency (mOTC-D), citrullinemia type 1 (CTLN1) and argininosuccinic aciduria (ASA) based on in vitro residual enzymatic activity for severity-adjustment. Results show that individuals with mOTC-D, CTLN1 and ASA are at risk of progressive linear growth impairment, recurrent annual HAEs and an unfavorable neurocognitive outcome despite being under long-term nitrogen scavenging pharmacotherapy. No overall superiority among existing nitrogen scavenging agents with regard to the individual's metabolic stability, linear growth impairment and poor neurocognitive outcome was observed. Novel therapeutic strategies are urgently needed to ultimately improve health outcomes in individuals with UCDs in order to sufficiently meet guideline-specific goals.
2026-03-11 | Looking Beyond Severe Hypertriglyceridemia when Diagnosing Adult-Onset Citrullinemia Type II.
Adult-onset citrullinemia type II (CTLN2) is a rare autosomal recessive urea cycle disorder caused by mutations in the solute carrier family 25 member 13 (SLC25A13) gene, which encodes citrin-a mitochondrial transporter involved in the malate-aspartate shuttle. In adults, CTLN2 may present atypically as isolated hypertriglyceridemia, often misattributed to secondary dyslipidaemia. A 30-year-old Vietnamese male with longstanding severe hypertriglyceridemia, first identified at age 13, was referred after an episode of acute pancreatitis at age 28. At presentation, plasma triglyceride levels reached 34.61 mmol/l. Secondary causes were excluded. Genetic testing via next-generation sequencing revealed compound heterozygous SLC25A13 mutations: c.2T>C and c.1638_1660dup, resulting in p.Met1Thr and p.Ala554GlyfsTer17, confirming the diagnosis of CTLN2. Initiation of a low-carbohydrate, high-protein, low-fat diet combined with fenofibrate (145 mg/day) led to a rapid reduction in triglyceride levels, normalizing within two weeks and remaining stable over three months. The treatment was well tolerated, with no reported adverse effects. CTLN2 should be considered in young adults with persistent, unexplained, and severe hypertriglyceridemia. Dietary modification constitutes the cornerstone of management, with fibrates playing a supportive role. Persistent, unexplained, and severe hypertriglyceridemia in young adults should prompt consideration of inherited metabolic disorders, including adult-onset citrullinemia type II (CTLN2).Genetic testing to detect compound heterozygous or homozygous SLC25A13 variants is essential for definitive diagnosis of CTLN2.In CTLN2, dietary management with a low-carbohydrate, high-protein, low-fat represents the cornerstone, with fibrates serving as adjunctive lipid-lowering therapy.
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
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.
gene therapies
2024-01-05 | Treatment and management for children with urea cycle disorder in chronic stage.
Urea cycle disorder (UCD) is a group of inherited metabolic diseases with high disability or fatality rate, which need long-term drug treatment and diet management. Except those with Citrin deficiency or liver transplantation, all pediatric patients require lifelong low protein diet with safe levels of protein intake and adequate energy and lipids supply for their corresponding age; supplementing essential amino acids and protein-free milk are also needed if necessary. The drugs for long-term use include nitrogen scavengers (sodium benzoate, sodium phenylbutyrate, glycerol phenylbutyrate), urea cycle activation/substrate supplementation agents (N-carbamylglutamate, arginine, citrulline), etc. Liver transplantation is recommended for pediatric patients not responding to standard diet and drug treatment, and those with severe progressive liver disease and/or recurrent metabolic decompensations. Gene therapy, stem cell therapy, enzyme therapy and other novel technologies may offer options for treatment in UCD patients. The regular biochemical assessments like blood ammonia, liver function and plasma amino acid profile are needed, and physical growth, intellectual development, nutritional intake should be also evaluated for adjusting treatment in time. 尿素循环障碍（UCD）是一组致死、致残率较高的遗传代谢病，需要长期饮食和药物治疗及管理。除希特林蛋白缺乏症和行肝移植治疗的患儿，其他慢性期患儿均需要终身低蛋白饮食，保证其相应年龄的安全蛋白质摄入量以及充足的碳水和脂肪的供能比，必要时补充必需氨基酸及无蛋白奶粉；药物治疗主要包括氮清除剂（苯甲酸钠、苯丁酸钠、苯丁酸甘油酯）、尿素循环激活/底物补充剂（N-氨基甲酰谷氨酸、精氨酸、瓜氨酸）等。规范饮食及药物治疗后未达预期效果、出现严重进展性肝病或出现反复发作的患儿建议行肝移植。基因疗法、干细胞疗法和酶替代疗法等新技术可能是UCD患儿治疗的新选择。UCD患儿需要定期检测血氨、肝功能和血氨基酸等生化指标，并评估体格生长、智力发育和营养摄入情况，及时调整治疗方案。. Urea cycle disorder (UCD) is a group of inherited metabolic diseases with high disability or fatality rate, which need long-term drug treatment and diet management. Except those with Citrin deficiency or liver transplantation, all pediatric patients require lifelong low protein diet with safe levels of protein intake and adequate energy and lipids supply for their corresponding age; supplementing essential amino acids and protein-free milk are also needed if necessary. The drugs for long-term use include nitrogen scavengers (sodium benzoate, sodium phenylbutyrate, glycerol phenylbutyrate), urea cycle activation/substrate supplementation agents (N-carbamylglutamate, arginine, citrulline), etc. Liver transplantation is recommended for pediatric patients not responding to standard diet and drug treatment, and those with severe progressive liver disease and/or recurrent metabolic decompensations. Gene therapy, stem cell therapy, enzyme therapy and other novel technologies may offer options for treatment in UCD patients. The regular biochemical assessments like blood ammonia, liver function and plasma amino acid profile are needed, and physical growth, intellectual development, nutritional intake should be also evaluated for adjusting treatment in time.
2023-09-25 | Lethality rescue and long-term amelioration of a citrullinemia type I mouse model by neonatal gene-targeting combined to SaCRISPR-Cas9.
Citrullinemia type I is a rare autosomal-recessive disorder caused by deficiency of argininosuccinate synthetase (ASS1). The clinical presentation includes the acute neonatal form, characterized by ammonia and citrulline accumulation in blood, which may lead to encephalopathy, coma, and death, and the milder late-onset form. Current treatments are unsatisfactory, and the only curative treatment is liver transplantation. We permanently modified the hepatocyte genome in lethal citrullinemia mice (Ass1fold/fold) by inserting the ASS1 cDNA into the albumin locus through the delivery of two AAV8 vectors carrying the donor DNA and the CRISPR-Cas9 platform. The neonatal treatment completely rescued mortality ensuring survival up to 5 months of age, with plasma citrulline levels significantly decreased, while plasma ammonia levels remained unchanged. In contrast, neonatal treatment with a liver-directed non-integrative AAV8-AAT-hASS1 vector failed to improve disease parameters. To model late-onset citrullinemia, we dosed postnatal day (P) 30 juvenile animals using the integrative approach, resulting in lifespan improvement and a minor reduction in disease markers. Conversely, treatment with the non-integrative vector completely rescued mortality, reducing plasma ammonia and citrulline to wild-type values. In summary, the integrative approach in neonates is effective, although further improvements are required to fully correct the phenotype. Non-integrative gene therapy application to juvenile mice ensures a stable and very efficient therapeutic effect.
2022-11-29 | Gene Therapy in Combination with Nitrogen Scavenger Pretreatment Corrects Biochemical and Behavioral Abnormalities of Infant Citrullinemia Type 1 Mice
Citrullinemia type I (CTLN1) is a rare autosomal recessive disorder caused by mutations in the gene encoding argininosuccinate synthetase 1 (ASS1) that catalyzes the third step of the urea cycle. CTLN1 patients suffer from impaired elimination of nitrogen, which leads to neurotoxic levels of circulating ammonia and urea cycle byproducts that may cause severe metabolic encephalopathy, death or irreversible brain damage. Standard of care (SOC) of CTLN1 consists of daily nitrogen-scavenger administration, but patients remain at risk of life-threatening decompensations. We evaluated the therapeutic efficacy of a recombinant adeno-associated viral vector carrying the ASS1 gene under the control of a liver-specific promoter (VTX-804). When administered to three-week-old CTLN1 mice, all the animals receiving VTX-804 in combination with SOC gained body weight normally, presented with a normalization of ammonia and reduction of citrulline levels in circulation, and 100% survived for 7 months. Similar to what has been observed in CTLN1 patients, CTLN1 mice showed several behavioral abnormalities such as anxiety, reduced welfare and impairment of innate behavior. Importantly, all clinical alterations were notably improved after treatment with VTX-804. This study demonstrates the potential of VTX-804 gene therapy for future clinical translation to CTLN1 patients.
2019-10-01 | Robust, Long-Term Culture of Endoderm-Derived Hepatic Organoids for Disease Modeling
Organoid technologies have become a powerful emerging tool to model liver diseases, for drug screening, and for personalized treatments. These applications are, however, limited in their capacity to generate functional hepatocytes in a reproducible and efficient manner. Here, we generated and characterized the hepatic organoid (eHEPO) culture system using human induced pluripotent stem cell (iPSC)-derived EpCAM-positive endodermal cells as an intermediate. eHEPOs can be produced within 2 weeks and expanded long term (>16 months) without any loss of differentiation capacity to mature hepatocytes. Starting from patient-specific iPSCs, we modeled citrullinemia type 1, a urea cycle disorder caused by mutations in the argininosuccinate synthetase (ASS1) enzyme. The disease-related ammonia accumulation phenotype in eHEPOs could be reversed by the overexpression of the wild-type ASS1 gene, which also indicated that this model is amenable to genetic manipulation. Thus, eHEPOs are excellent unlimited cell sources to generate functional hepatic organoids in a fast and efficient manner.
2019-06-19 | Insights into Gene Therapy for Urea Cycle Defects by Mathematical Modeling
Metabolic liver diseases are attractive gene therapy targets that necessitate reconstitution of enzymatic activity in functionally complex biochemical pathways. The levels of enzyme activity required in individual hepatocytes and the proportion of the hepatic cell mass that must be gene corrected for therapeutic benefit vary in a disease-dependent manner that is difficult to predict. While empirical evaluation is inevitably required, useful insights can nevertheless be gained from knowledge of disease pathophysiology and theoretical approaches such as mathematical modeling. Urea cycle defects provide an excellent example. Building on a previously described one-compartment model of the urea cycle, we have constructed a two-compartment model that can simulate liver-targeted gene therapy interventions using the computational program Mathematica. The model predicts that therapeutically effective reconstitution of ureagenesis will correlate most strongly with the proportion of the hepatic cell mass transduced rather than the level of enzyme-encoding transgene expression achieved in individual hepatocytes. Importantly, these predictions are supported by experimental data in mice and human genotype/phenotype correlations. The most notable example of the latter is ornithine transcarbamylase deficiency (X-linked) where impairment of ureagenesis in male and female patients is closely simulated by the one- and two-compartment models, respectively. Collectively, these observations support the practical value of mathematical modeling in evaluation of the disease-specific gene transfer challenges posed by complex metabolic phenotypes.
cell therapies
2026-06-04 | Sudden Acute Liver Failure in Citrullinemia Type 1: An Argument for Earlier Liver Transplantation?
Acute liver failure has been described at initial presentation of citrullinemia type 1, but recurrent hepatic decompensations during medical treatment have not been reported. We describe 5 children developing episodes of acute liver failure, including one who required emergency liver transplantation. Our findings expand the citrullinemia type 1 phenotype and support early liver transplant referral in selected cases.
2025-05-12 | Supplementation with Kluyveromyces marxianus probiotic yeasts in sows and their piglets in early post- weaning: effects in biological and productive parameters
Weaning in pig production is a stressful event that impacts intestinal biology and increases the risk of infections, impairing piglet development and leading to economic losses. Antibiotic preventive administration has been used to improve the productive parameters, but nowadays this practice has been banned because of its contribution to antimicrobial resistance generation. So, alternatives are urgently needed. Kluyveromyces marxianus CIDCA 9121 has immunomodulatory and convenient biotechnological properties and was studied for preventing post-weaning stress, for which 8 sows were supplemented with 109 CFU per kg of feed from 20 days before piglets´ birth until weaning. Then, piglets were supplemented with 2x109 CFU/ kg of feed for 20 days more. A control group of 8 sows and their piglets not supplemented with yeast was included. Productive parameters were registered from birth to 60 days of age. Five days post-weaning, 5 animals from each group were euthanized; small intestines and blood were taken for histopathology analysis and amino acid analysis respectively. Intestinal content was sampled to determine the main bacterial groups by qPCR. Piglets born from supplemented sows (n = 132) weighed more than controls (n = 130; p < 0,05). During farrowing, there were no differences among groups but 40 days after weaning the supplemented group weighted 10% more than the control group (p < 0,05). We found an increase in citrullinemia and in the small intestine mucosal thickness in supplemented piglets post-weaning indicating better tolerance to post-weaning stress. Our results suggest that K. marxianus CIDCA 9121 is a good candidate for supplementation of gestating sows and postweaning piglets.
2024-11-19 | Development of optimised human iPSC-derived hepatocytes with improved liver function for in vitro metabolic disease modelling and toxicity studies
Abstract Background & Aims Liver disease is a rising cause of mortality worldwide. Primary human hepatocytes (PHH) and hepatocellular cancer cells are currently used in drug development, however, they come with limitations, including limited supply, rapid loss of function, and tumorigenic origin. In addition, current iPSC differentiation protocols lead to the generation of hepatocyte-like cells with compromised liver-related features. We hypothesised that optimisation of iPSC differentiation protocols can lead to the generation of hepatocyte-like cells with improved metabolic functionality for disease modelling and toxicity screening studies. Methods Healthy human iPSCs were differentiated to hepatocyte-like cells (Opti-HEP) using a novel 3-step differentiation protocol. Hepatocyte functionality was assessed, including liver maturity marker expression, urea synthesis, de novo gluconeogenesis, and CYP450 expression, activity, and induction. Suitability of Opti-HEP to predict drug-induced liver injury (DILI) was evaluated by cell viability assays. CRISPR/Cas9 gene editing was employed to generate in vitro inherited metabolic disease models. Results Opti-HEP expressed similar liver maturity marker levels to those seen in primary human hepatocytes (PHH), in addition to functional urea and gluconeogenesis pathways. CYP450 expression and activity were comparable between Opti-HEP and PHH, with both cell types showing similar levels of CYP3A4 induction upon 1α,25-hydroxy-vitamin D3 treatment. Opti-HEP accurately predicted DILI, following treatment with 7 drugs of known DILI liability. CRISPR-derived Opti-HEP harbouring mutations for inherited metabolic disorders (Ornithine Transcarbamylase Deficiency, Progressive Familial Intrahepatic Cholestasis Type 2, Citrullinemia Type 1) recapitulated key pathophysiological disease features, including reduced protein expression, impaired urea secretion, and bile acid transport. Conclusions We demonstrate the generation of optimised iPSC-derived hepatocytes with enhanced liver functionality that is comparable to PHH. These data alongside the expansion capacity and amenability of these cells highlight the opportunities this model can offer in the space of disease modelling and large-scale drug efficacy and hepatotoxicity screening.
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.
2022-09-26 | Modelling urea cycle disorders using iPSCs
Abstract The urea cycle is a liver-based pathway enabling disposal of nitrogen waste. Urea cycle disorders (UCDs) are inherited metabolic diseases caused by deficiency of enzymes or transporters involved in the urea cycle and have a prevalence of 1:35,000 live births. Patients present recurrent acute hyperammonaemia, which causes high rate of death and neurological sequelae. Long-term therapy relies on a protein-restricted diet and ammonia scavenger drugs. Currently, liver transplantation is the only cure. Hence, high unmet needs require the identification of effective methods to model these diseases to generate innovative therapeutics. Advances in both induced pluripotent stem cells (iPSCs) and genome editing technologies have provided an invaluable opportunity to model patient-specific phenotypes in vitro by creating patients’ avatar models, to investigate the pathophysiology, uncover novel therapeutic targets and provide a platform for drug discovery. This review summarises the progress made thus far in generating 2- and 3-dimensional iPSCs models for UCDs, the challenges encountered and how iPSCs offer future avenues for innovation in developing the next-generation of therapies for UCDs.
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.
2025-02-21 | Developing splice-switching oligonucleotides for urea cycle disorder using an integrated diagnostic and therapeutic platform.
Citrin deficiency (CD) is an autosomal recessive urea cycle disorder caused by biallelic loss-of-function variants in the SLC25A13 gene, leading to life-threatening hyperammonemia and hypoglycemia. Variants in deep introns can cause genetic diseases by altering splicing and are often missed by current diagnostic tools. Splice-switching oligonucleotides (SSOs) can resolve certain intronic variants, but patients harboring such variants need to be identified. We present a lean workflow from molecular diagnostics to SSO development to resolve splice-altering variants in deep introns that is applicable to other genetic disorders. A deep intronic-gene panel was designed to identify deep intronic variants. SSOs were then developed and validated in vitro using a minigene assay and induced hepatocytes, and target engagement was verified in vivo by hydrodynamic tail vein injection of minigenes and SSOs. With the deep intronic-gene panel and RNA analysis, we identified a novel SLC25A13 c.469-2922G>T variant that promotes the inclusion of a premature stop codon-containing pseudo-exon, SLC25A13-PE5, thereby causing CD. Using a stepwise rational SSO design approach, we identified potent candidates inhibiting SLC25A13-PE5 at EC50 <2 nM in vitro. Upon conjugating the SSOs with GalNAc (N-acetylgalactosamine), they were validated to rescue normal protein expression and restore ureagenesis and ammonia clearance, key urea cycle functions, in patient-derived induced hepatocytes. In vivo on-target efficacy of the clinical GalNAc-SSO candidate, in the absence of acute toxicity and inflammation, was observed in a mouse model with exogenous hepatic minigene expression. Our data validates a platform to redefine the molecular diagnosis of urea cycle disorders and provides proof-of-concept for a precision therapy for patients with CD, for whom the only effective treatment is liver transplantation. Deep intronic variants are common causes of genetic diseases that are commonly neglected. In this study, we demonstrate an integrated precision diagnostic and therapeutic approach for urea cycle disorders. Specifically, we focus on citrin deficiency, going from the discovery of a novel splice variant in the SLC25A13 gene with our novel deep intronic-gene panel for urea cycle disorders, to the development and in vivo validation of an efficacious splice-switching oligonucleotide candidate for the pathogenic splice variant. We envision the possibility of extrapolating this pipeline to the diagnosis and development of treatments for other rare genetic 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.
2024-07-18 | The therapeutic landscape of citrin deficiency.
Citrin deficiency (CD) is a recessive, liver disease caused by sequence variants in the SLC25A13 gene encoding a mitochondrial aspartate-glutamate transporter. CD manifests as different age-dependent phenotypes and affects crucial hepatic metabolic pathways including malate-aspartate-shuttle, glycolysis, gluconeogenesis, de novo lipogenesis and the tricarboxylic acid and urea cycles. Although the exact pathophysiology of CD remains unclear, impaired use of glucose and fatty acids as energy sources due to NADH shuttle defects and PPARα downregulation, respectively, indicates evident energy deficit in CD hepatocytes. The present review summarizes current trends on available and potential treatments for CD. Baseline recommendation for CD patients is dietary management, often already present as a self-selected food preference, that includes protein and fat-rich food, and avoidance of excess carbohydrates. At present, liver transplantation remains the sole curative option for severe CD cases. Our extensive literature review indicated medium-chain triglycerides (MCT) as the most widely used CD treatment in all age groups. MCT can effectively improve symptoms across disease phenotypes by rapidly supplying energy to the liver, restoring redox balance and inducing lipogenesis. In contrast, sodium pyruvate restored glycolysis and displayed initial preclinical promise, with however limited efficacy in adult CD patients. Ursodeoxycholic acid, nitrogen scavengers and L-arginine treatments effectively address specific pathophysiological aspects such as cholestasis and hyperammonemia and are commonly administered in combination with other drugs. Finally, future possibilities including restoring redox balance, amino acid supplementation, enhancing bioenergetics, improving ureagenesis and mRNA/DNA-based gene therapy are also discussed.
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.
other
2026-06-17 |
Twist variant codon usage for yeast.
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 2,193 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. Under the current ACMG guidelines, inclusion of our data yielded definitive classifications (pathogenic or likely pathogenic) for all 25 ClinVar VUS falling in the functionally impaired range of our assay. 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.
2023-03-16 | Exogenous aralar/slc25a12 can replace citrin/slc25a13 as malate aspartate shuttle component in liver
The deficiency of CITRIN, the liver mitochondrial aspartate–glutamate carrier (AGC), is the cause of four human clinical phenotypes, neonatal intrahepatic cholestasis caused by CITRIN deficiency (NICCD), silent period, failure to thrive and dyslipidemia caused by CITRIN deficiency (FTTDCD), and citrullinemia type II (CTLN2). Clinical symptoms can be traced back to disruption of the malate-aspartate shuttle due to the lack of citrin. A potential therapy for this condition is the expression of aralar, the AGC present in brain, to replace citrin. To explore this possibility we have first verified that the NADH/NAD+ ratio increases in hepatocytes from citrin(−/−) mice, and then found that exogenous aralar expression reversed the increase in NADH/NAD+ observed in these cells. Liver mitochondria from citrin (−/−) mice expressing liver specific transgenic aralar had a small (~ 4–6 nmoles x mg prot−1 x min−1) but consistent increase in malate aspartate shuttle (MAS) activity over that of citrin(−/−) mice. These results support the functional replacement between AGCs in the liver. To explore the significance of AGC replacement in human therapy we studied the relative levels of citrin and aralar in mouse and human liver through absolute quantification proteomics. We report that mouse liver has relatively high aralar levels (citrin/aralar molar ratio of 7.8), whereas human liver is virtually devoid of aralar (CITRIN/ARALAR ratio of 397). This large difference in endogenous aralar levels partly explains the high residual MAS activity in liver of citrin(−/−) mice and why they fail to recapitulate the human disease, but supports the benefit of increasing aralar expression to improve the redox balance capacity of human liver, as an effective therapy for CITRIN deficiency.
2012-01-20 | Substrate Specificity of the Two Mitochondrial Ornithine Carriers Can Be Swapped by Single Mutation in Substrate Binding Site
Mitochondrial carriers are a large family of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has indicated that these transporters have substrate binding sites in a similar location of the central cavity consisting of three major contact points. Here we have characterized mutations of the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 by carrying out transport assays with a set of different substrates. The different substrate specificities of the two isoforms, which share 87% identical amino acids, were essentially swapped by exchanging a single residue located at position 179 that is arginine in ORC1 and glutamine in ORC2. Altogether the substrate specificity changes demonstrate that Arg-179 and Glu-180 of contact point II bind the Cα carboxylate and amino group of the substrates, respectively. Residue Glu-77 of contact point I most likely interacts with the terminal amino group of the substrate side chain. Furthermore, it is likely that all three contact points are involved in the substrate-induced conformational changes required for substrate translocation because Arg-179 is probably connected with Arg-275 of contact point III through Trp-224 by cation-π interactions. Mutations at position 179 also affected the turnover number of the ornithine carrier severely, implying that substrate binding to residue 179 is a rate-limiting step of the catalytic transport cycle. Given that Arg-179 is located in the vicinity of the matrix gate, it is concluded that it is a key residue in the opening of the carrier to the matrix side. Mitochondrial carriers are a large family of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has indicated that these transporters have substrate binding sites in a similar location of the central cavity consisting of three major contact points. Here we have characterized mutations of the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 by carrying out transport assays with a set of different substrates. The different substrate specificities of the two isoforms, which share 87% identical amino acids, were essentially swapped by exchanging a single residue located at position 179 that is arginine in ORC1 and glutamine in ORC2. Altogether the substrate specificity changes demonstrate that Arg-179 and Glu-180 of contact point II bind the Cα carboxylate and amino group of the substrates, respectively. Residue Glu-77 of contact point I most likely interacts with the terminal amino group of the substrate side chain. Furthermore, it is likely that all three contact points are involved in the substrate-induced conformational changes required for substrate translocation because Arg-179 is probably connected with Arg-275 of contact point III through Trp-224 by cation-π interactions. Mutations at position 179 also affected the turnover number of the ornithine carrier severely, implying that substrate binding to residue 179 is a rate-limiting step of the catalytic transport cycle. Given that Arg-179 is located in the vicinity of the matrix gate, it is concluded that it is a key residue in the opening of the carrier to the matrix side.
Access all drug discovery papers and probability of success in trials forecasts:
Access all drug discovery papers and probability of success in trials forecasts:
Drug Discovery Landscape
2 orphan drug designations for Citrullinemia.
2 orphan drug designations for Citrullinemia.
Drug | Therapy type | Regulator | Orphan designation | Approval | Sponsor |
|---|---|---|---|---|---|
Sodium benzoate | small molecules | EMA | 2016-07-14 | — | Lucane Pharma SA |
Heterologous human adult liver-derived progenitor cells | cell therapies | EMA | 2013-07-17 | — | Cellaion |
Let's accelerate rare disease drug discovery
Let's accelerate drug discovery
Get access to Explority AI’s forecasts to outperform average preclinical success rates. Whether you’re expanding your R&D pipeline, evaluating a partnership, or simply have a question — we’d love to hear from you.