Urea-Cycle Disorders As a Paradigm for Inborn Errors of Hepatocyte Metabolism

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42 Palakurthi, S.S. et al. (2000) Inhibition of 49 Harigai, M. et al. (1996) A cis-acting element in 56 Raught, B. et al. (1996) Expression of a translation initiation mediates the anticancer the BCL-2 gene controls expression through translationally regulated, dominant-negative effect of the n-3 polyunsaturated fatty acid translational mechanisms. Oncogene CCAAT/enhancer-binding protein β isoform and eicosapentaenoic acid. Cancer Res. 60, 2919–2925 12, 1369–1374 up-regulation of the eukaryotic translation 43 Palakurthi, S.S. et al. (2001) Anticancer effects of 50 Calkhoven, C.F. et al. (2000) Translational control initiation factor 2α are correlated with neoplastic thiazolidinediones are independent of peroxisome of C/EBPα and C/EBPβ isoform expression. transformation of mammary epithelial cells. proliferator-activated receptor γ and mediated by Genes Dev. 14, 1920–1932 Cancer Res. 56, 4382–4386 inhibition of translation initiation. Cancer Res. 51 Harding, H.P. et al. (2000) Regulated translation 57 Rask, K. et al. (2000) Increased expression of the 61, 6213–6218 initiation controls stress-induced gene expression transcription factors CCAAT-enhancer binding 44 Aktas, H. et al. (1998) Depletion of intracellular in mammalian cells. Mol. Cell 6, 1099–1108 protein-β (C/EBPβ) and C/EBPζ (CHOP) correlate Ca2+ stores, phosphorylation of eIF2α, and 52 Descombes, P. and Schibler, U. (1991) A with invasiveness of human colorectal cancer. sustained inhibition of translation initiation liver-enriched transcriptional activator protein, Int. J. Cancer 86, 337–343 mediate the anticancer effects of clotrimazole. LAP, and a transcriptional inhibitory protein, LIP, 58 Hidalgo, M. and Rowinsky, E.K. (2000) The Proc. Natl. Acad. Sci. U. S. A. 95, 8280–8285 are translated from the same mRNA. Cell rapamycin-sensitive signal transduction pathway 45 Kozak, M. (1991) An analysis of vertebrate mRNA 67, 569–579 as a target for cancer therapy. Oncogene sequences: intimations of translational control. 53 Ossipow, V. et al. (1993) CCAAT/enhancer-binding 19, 6680–6686 J. Cell Biol. 115, 887–903 protein mRNA is translated into multiple 59 Neshat, M.S. et al. (2001) Enhanced sensitivity of 46 Hengst, L. and Reed, S.I. (1996) Translational proteins with different transcription activation PTEN-deficient tumors to inhibition of control of p27kip1 accumulation during the cell potentials. Proc. Natl. Acad. Sci. U. S. A. FRAP/mTOR. Proc. Natl. Acad. Sci. U. S. A. cycle. Science 271, 1861–1864 90, 8219–8223 98, 10314–10319 47 Rousseau, D. et al. (1996) Translation initiation of 54 Lin, F.T. et al. (1993) A 30-kDa alternative 60 Louro, I.D. et al. (1999) The zinc finger protein ornithine decarboxilase and nucleocytoplasmic translation product of the CCAAT/enhancer GLI induces cellular sensitivity to the mTOR transport of cyclin D1 mRNA are increased in binding protein α message: transcriptional inhibitor rapamycin. Cell Growth Differ. cells overexpressing eukaryotic initiation factor activator lacking antimitotic activity. Proc. Natl. 10, 503–516 4E. Proc. Natl. Acad. Sci. U. S. A. 93, 1065–1070 Acad. Sci. U. S. A. 90, 9606–9610 61 Guba, M. et al. (2002) Rapamycin inhibits 48 Ghilardi, N. et al. (1998) Thrombopoietin 55 Kowenz-Leutz, E. and Leutz, A. (1999) A C/EBPβ primary and metastatic tumor growth by production is inhibited by a translational isoform recruits the SWI/SNF complex to activate antiangiogenesis: involvement of vascular mechanism. Blood 92, 4023–4030 myeloid genes. Mol. Cell 4, 735–743 endothelial growth factor. Nat. Med. 8, 128–135

Urea-cycle disorders as a paradigm for inborn errors of hepatocyte metabolism

Asad Mian and Brendan Lee

Urea-cycle disorders (UCDs) are a group of inborn errors of hepatocyte including (but not limited to) organic acidemias, metabolism that are caused by the loss of enzymes involved in the process of fatty-acid-oxidation disorders and amino transferring nitrogen from ammonia to urea, via the urea cycle (UC). Recent acidopathies. UCDs are cell-autonomous disease genetic analyses of inherited disorders that present with hyperammonemia processes and, therefore, a cure requires the demonstrate the function of cellular transporters that regulate the availability correction of significant numbers of hepatocytes. of UC intermediates. The regulation of UC intermediates, such as arginine, These disorders also represent a unique genetic could have far reaching implications on nitric-oxide synthesis and vascular model for understanding how gene–environment tone. Hence, each UCD and UC-related disorder constitutes a unique interactions affect nitrogen homeostasis, which gene-nutrient interaction that is crucial for postnatal homeostasis. Recent is affected by the intake of both energy molecules advances in the diagnosis and management of UCDs include the application of and protein. The recent discoveries of several in vivo metabolic-flux measurements. Cumulative morbidity is still high cellular transporters show how the availability despite dietary and pharmacological therapies and, hence, both cell and gene of urea cycle (UC) intermediates further impacts therapies are being pursued as possible long-term corrective treatments. these processes. Although gene-replacement therapy has suffered recent clinical setbacks, new vector developments offer hope for the treatment of cell-autonomous Urea cycle: pathway to phenotype defects of hepatocyte metabolism. The UC is the only metabolic pathway capable of disposing of excess nitrogen. It converts nitrogen, Urea-cycle disorders (UCDs) are seen as models of derived from dietary protein intake (i.e. enteral classic inborn errors of hepatocyte metabolism. sources) and the breakdown of endogenous protein Hence, new developments in their diagnosis and (i.e. peripheral sources), into urea, which is treatment are relevant to a host of other disorders, water-soluble and easily excreted from the body (Fig. 1).

Fig. 1. The flow of nitrogen intermediates in the urea Ornithine cycle (UC). The UC converts nitrogen, derived Extracellular space from dietary protein intake and the breakdown Diet and + Ð NH4 HCO3 of endogenous protein catabolism (catabolism), into urea, which can be excreted from the body. The steps + Ð of this cycle, and the six NH4 + HCO3 enzymes involved, are indicated in red. The Glutamate NAGS Ornithine dashed red arrow depicts + NAG CPS-I Ornithine Urea the overflow of excess Acetyl CoA carbamyl phosphate (CP) OTC ARG1 into pyrimidine synthesis CP and, hence, to orotic acid, Mitochondrion which is excreted in the Arginine urine. The dashed green Citrulline Arginine arrow depicts the NOS generation of nitric oxide Aspartate (NO) when arginine is converted into citrulline by nitric oxide synthase NO ASL (NOS). The green cylinders Orotic acid Citrulline denote the ornithine and citrulline transporter (ORNT1; SLC25A15), Aspartate ASS and the blue cylinders represent the cationic- Argininosuccinate amino-acid transporter found on intestinal- and Cytoplasm kidney-epithelial cells (SLC7A7). The purple cylinder denotes citrin, a TRENDS in Molecular Medicine mitochondrial aspartate and glutamate transporter (SLC25A13). Two moles of nitrogen (one from ammonia and one catabolism, although protein intake through feeding Abbreviations: ARG1, arginase; ASL, from aspartate) are transferred into two moles also contributes. Initial signs include somnolence argininosuccinate lyase; of urea and a deficiency in any of the six classical and poor feeding, and these are usually followed by ASS, argininosuccinate enzymes of the pathway causes a UCD (Table 1). vomiting, lethargy and coma (Box 1). A common early synthetase; CPS-I, carbamyl-phosphate- With the exception for argininemia, which often symptom in newborns with hyperammonemia is synthetase-I; NAG, presents with spastic diplegia, the neonatal central hyperventilation, which is thought to result N-acetyl-glutamate; NAGS, presentation of UCDs is characterized by from cerebral edema caused by accumulation of N-acetyl-glutamate hyperammonemia, encephalopathy and respiratory ammonia and other metabolites, and leads to synthase; OTC, ornithine transcarbamylase. alkalosis. However, disorders associated with partial respiratory alkalosis [5]. Progressive cerebral activity of the urea cycle can present late, with edema ultimately leads to abnormal posture and pleiotropic manifestations ranging from normal encephalopathy, with hypoventilation and respiratory development with acute metabolic decompensation, arrest. In addition, approximately 50% of infants to psychiatric illness (Box 1). Rapid diagnosis and with severe hyperammonemia have seizures [5]. treatment are crucial in preventing neurological Hyperammonemia can be chronic or acute, the acute injury [1,2] and it is generally accepted that the form resulting from metabolic decompensation duration, frequency and degree of hyperammonemia associated with stressors, such as viral illness or contribute to cumulative neurological morbidity. childbirth [5]. In patients with partial urea-cycle- The overall frequency of UCDs is approximately enzyme deficiencies, symptoms do not present until one per 30 000 live births [3]. They all have an after the newborn period. This delay is most common Asad Mian Dept of Molecular and autosomal recessive inheritance pattern, with the with partial OTCD, but it can also occur with other Human Genetics, Baylor exception of ornithine-transcarbamylase deficiency UCDs [6]. These children suffer from chronic College of Medicine, (OTCD), which is X-linked. The clinical severity of vomiting, developmental delay, seizures and Houston, TX 77030, USA. OTCD in females depends upon the pattern of psychiatric illness (Box 1). *Brendan Lee X-inactivation in the liver, and ranges from The pathogenesis of any inborn error of Dept. of Molecular and asymptomatic to almost as severe as in affected metabolism can be attributed to either the primary or Human Genetics and Howard Hughes Medical males [4]. Newborns with UCDs typically appear well secondary effects of the accumulation of metabolites Institute, Baylor College for the first 24–48 h after birth, because the placental upstream of the block and/or a deficiency of essential of Medicine, Houston, unit compensates for the deficiency in utero. Hence, nutrients downstream of the block. Both types of TX 77030, USA. *e-mail: blee@ symptoms appear in the first few days after birth, effects are evident in UCD patients. The immediate bcm.tmc.edu resulting primarily from endogenous protein cerebral toxicity can be attributed to the accumulation

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Table 1. Biochemical and genetic characteristics of the enzymes of the urea cycle and their deficiency states

Enzyme Tissue distribution Chromosomal Molecular Deficiency state Animal model location complex

N-acetylglutamate synthetase (NAGS) Liver, gut, kidney 17q21.31 Monomer NAGS deficiency Carbamyl-phosphate-synthetase-I (CPS-I) Liver, gut, kidney 2q35 Dimer CPS-I deficiency Murine Ornithine transcarbamylase (OTC) Liver, gut, kidney Xp21.1 Trimer OTC deficiency Murine Argininosuccinate synthetase Liver, kidney, fibroblasts 9q34 Tetramer Citrullinemia Bovine and murine Argininosuccinate lyase Liver, kidney, fibroblasts, brain 7cen-q11.2 Tetramer Argininosuccinic aciduria Murine Arginase Liver, kidney, erythrocytes 6q23 Trimer Argininemia Murine

of ammonia: acute hyperammonemia is thought to are found in argininosuccinic-acid-synthetase cause an imbalance in extracellular and intracellular deficiency (ASSD), elevated argininosuccinic acid glutamate concentrations, and an associated activation is characteristic of argininiosuccinic-acid-lyase of N-methyl-D-aspartate-type (NMDA) glutamate deficiency (ASLD), and raised arginine levels are receptors [7], which is thought to lead to excitotoxic found in arginase deficiency. Enzymatic testing on neurotoxicity and cerebral edema. In the longer term, fibroblasts, erythrocytes and liver is also possible a deficiency of semi-essential amino acids, such as for some of the conditions. Partial-activity cases of arginine, contributes to a failure to thrive. Importantly, OTCD can be diagnosed by provocative testing even conventional therapeutic interventions (both using allopurinol challenge, which increases orotic pharmacological and dietary) carry a risk of leading aciduria as a result of an increased flux of cytosolic to deficiencies in essential amino acids, such as those carbamyl phosphate into pyrimidine synthesis with branched chains. Catabolic stresses, including (Fig. 1) [10,11]. DNA mutation detection is clinically amino-acid deficiency, cause a mobilization of available only for OTCD. In OTCD (and probably peripheral nitrogen stores (primarily glutamine the other autosomal recessive conditions), the and alanine), increasing total nitrogen flux neonatal-onset patients have mutations that and thereby placing the patient at risk of completely abolish enzyme activity, whereas hyperammonemia. The burst of inflammatory late-onset sufferers show varying degrees of cytokines (e.g. interleukin-6) associated with partial enzyme deficiency [6]. acute illness has similar effects on the total nitrogen flux and, hence, increases the risk of metabolic Metabolic flux correlates with phenotype decompensation and hyperammonemia [8]. The limitation of conventional approaches to diagnosis is that they do not directly measure the Biochemical and molecular diagnosis of UCDs block. In fact, even individual genotypes might not be Conventional approach to diagnosis predictive of phenotypic severity [12]. Diagnosis of The diagnostic hallmark of any inborn metabolic neonatal-onset UCDs is generally straightforward, as error is the accumulation of intermediary metabolites a result of null activity of the UC. However, clinical upstream of the block. In null-activity cases that and laboratory diagnosis of partial deficiencies is present in the neonatal period diagnosis is relatively difficult, especially during asymptomatic periods. straightforward. However, it can be more difficult in In these cases, linking phenotypic severity with partial-activity cases. This is because intermittent genotype and/or in vitro enzyme activity is often plasma samplings give non-steady-state concentrations imprecise [13,14]: correlating phenotype with in the blood and might not adequately represent the genotype is difficult because both subtle (nonsense rate of production and elimination of the metabolite. In theory, the metabolic flux through a pathway Box 1. Clinical symptoms of urea-cycle disorders (i.e. the rate of appearance and disappearance) directly correlates with phenotypic and genotypic Neonatal-onset severity, but measurement of flux has proved to be Somnolence impractical for many disorders. Poor feeding Vomiting The diagnosis of severe neonatal UCDs has long Lethargy been dependent on the measurement of non-steady- Seizures state accumulations of intermediary metabolites, Hyperammonemic coma particularly ammonia, upstream of the block [9]. Late-onset Quantifying plasma levels of different metabolites of Chronic vomiting the urea cycle is also helpful in differentiating among Developmental delay UCDs [5,9]. For example, elevated levels of ammonia Seizures Psychiatric illness and glutamine are found in all disorders except Postpartum decompensation argininemia, elevated orotic acid and low citrulline Hyperammonemia associated with valproate therapy levels are a feature of OTCD, raised levels of citrulline

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Fig. 2. Metabolic flux Therapies for UCDs through the urea cycle as 0.6 Conventional metabolic management measured by the transfer Control subjects of 15N from glutamine to Heterozygous OTCD females The conventional approach to the treatment of 15N in urea. The ratio of 0.5 ASSD and ASLD heterozygotes inborn errors of metabolism involves the following [15N]urea:[15N]glutamine Males with partial OTCD basic principles: (1) restriction of upstream essential distinguishes control 0.4 Null patients subjects (filled circles) nutrients to prevent intoxication, while providing from null patients with enough to sustain growth and development; 0.3 urea-cycle disorders N] glutamine (2) supplementation of downstream nutrients to (neonatal or late 15 prevent secondary deficiency; and (3) stimulation of presentation) (open 0.2 circles), irrespective of alternative routes of disposal of precursor metabolites. enzymatic defect. It can The treatment of UCDs is a model for many inborn N] urea: [ N] urea: 0.1

also distinguish 15

[ errors and encompasses all three of these approaches: asymptomatic restriction of dietary protein intake, aggressive heterozygous carriers of 0.0 ‘argininosuccinic-acid- support during periods of catabolic stress, and synthetase deficiency’ Ð0.1 pharmacotherapy with sodium phenylbutyrate and ‘argininiosuccinic- TRENDS in Molecular Medicine (Buphenyl). In the body, phenylbutyrate is converted acid-lyase deficiency’ (filled triangles), to phenylacetate, which conjugates with glutamine to male patients with form phenylacetylglutamine (PAG). PAG is efficiently partial ornithine- and missense mutations) changes and large excreted in the urine, thus providing an alternative transcarbamylase rearrangements can cause the loss of function route for nitrogen disposal [17]. deficiency (OTCD) (upside- down filled triangles) and of a multimeric protein complex, and correlating Citrulline and arginine are given to fulfill heterozygous females phenotype with in vitro enzyme activity can be nutritional requirements and to provide precursors with OTCD (filled squares) inaccurate because many in vitro biochemical assays for enzymatic steps upstream of the putative block. from control subjects. Reproduced, with are performed in non-physiological conditions of Although dietary regulation and pharmacotherapy permission, from [16]. substrate excess. can decrease long-term morbidity in some cases, Recently, in a variety of UC enzymatic deficiencies, especially in females with partial OTCD, the calculations of in vivo total-body urea-synthesis rates prognosis for neonatally presenting null-activity and urea-cycle-specific nitrogen flux, determined patients is still very poor, owing to recurrent using stable isotopes, have shown an excellent hyperammonemia secondary to catabolic stresses, correlation with both the phenotypic severity in such as viral illness [18–20]. Interestingly, recent patients and the carrier status of the parents. In these stable-isotope studies have shown that ammonia approaches, a precursor pool of nitrogen is enriched from enteral sources of nitrogen is more readily 15 15 with the stable isotope, N. Both NH4Cl and converted to urea than is nitrogen from peripheral [15N]glutamine have been used, in different sources (e.g. from stress-induced mobilization) [21]. studies [15,16]. In the [15N]glutamine study, the ratio This supports the idea of a direct correlation of isotopic enrichment of [15N]urea:[15N]glutamine between episodes of catabolic stress and an distinguished control subjects from UCD patients increased risk of hyperammonemia. Hence, for with neonatal or late presentations, irrespective of severe neonatal OTCD, and also for carbamyl- enzymatic defect (Fig. 2) [16]. It also distinguished phosphate-synthetase-I (CPS-I) deficiency, both of asymptomatic heterozygous carriers of ASSD and which have a poor long-term prognosis, alternative ASLD, and patients with partial OTCD, from control therapies are being tested [22]. subjects. Recently, this approach has supplemented the complex risk–benefit considerations of liver Cell and gene therapies transplantation (cell therapy) as a treatment for UCDs can be treated in the long term by correction of partial OTCD [4]. the enzymatic defect in hepatocytes. Orthotopic Measurement of the metabolic flux of a labeled liver transplantation is currently the primary precursor through a dysregulated biochemical method of achieving this, and recent advances in pathway is the best phenotypic marker of clinical organ procurement, surgical technique (e.g. in situ severity. The key question is whether the technique split harvesting of livers) and immunosuppression can be used widely without sacrificing the have significantly decreased morbidity and uniformity of results. Furthermore, in many mortality [22–24]. However, short-term complications metabolic studies, a steady-state pool of precursors with surgery, and long-term problems associated is required, necessitating regulated protein with chronic immunosuppression (e.g. Epstein–Barr- intake for several days. In addition, the gas virus-associated lymphoproliferative disease), have chromatography/mass spectrometry technology spurred continued efforts to develop gene-replacement is not always readily available in different therapies, initially as a short-term measure until centers. These problems represent significant liver transplantation can be performed but, obstacles to the widespread application of this ultimately, for long-term correction of the defect [22]. approach to the diagnosis and management of Significant morbidity and mortality are associated inborn errors of metabolism. with neonatal UCDs and, hence, the risk–benefit

http://tmm.trends.com Review TRENDS in Molecular Medicine Vol.8 No.12 December 2002 587 considerations make gene-replacement therapy a patients with partial OTCD. One patient treated with viable alternative to surgery for long-term correction 6 × 1011 viral particles/kg suffered significant acute of these disorders. Indeed, the UCDs have been a toxicity, resulting in death. It is likely that this subject model for the development of human gene-replacement had suffered systemic-inflammatory-response therapy for cell-autonomous disorders of hepatocyte syndrome, with disseminated intravascular metabolism. So far, adenoviral vectors have offered coagulation [22,34]. The dose at which this adverse the best prospects for obtaining a clinically relevant event occurred was lower than that which caused effect, owing to their unique hepatotropism after a similar toxicities in non-human primates, indicating single intravenous injection. The cell-autonomous that there is a significant pharmacogenetic variation nature of the enzymatic defects also dictates a in the acute response to adenoviral-vector delivery in requirement for efficient hepatocyte transduction and humans [35]. In the other patients, adverse effects high-level gene expression. Based on clinical data and included a flu-like episode, transient elevations in animal models, appreciable therapeutic correction of temperature and of hepatic transaminases, and at least 15–30% of hepatocytes is required for clinical dose-related thrombocytopenia [34]. Unfortunately, effect, further suggesting the need for an efficient no significant metabolic correction was observed. adenoviral gene-delivery system [25,26]. Most This study again suggests that there are significant preclinical studies in animal models have focused on species differences in the pharmacological response OTCD and ASSD. Correction of the metabolic to therapies of this kind. Furthermore, it highlights phenotype has been demonstrated in the ‘sparse fur’ the importance of improving the therapeutic mouse model of partial OTCD, using adenoviral index for gene therapies, to safely accommodate vectors in which the E1, or E1 and E2, early genes pharmacogenetic variation in the human population. were deleted [25]. In a bovine model of citrullinemia A recent development that has significantly (ASSD), treatment with an E1-deleted adenoviral improved the therapeutic index of adenoviral gene vector expressing human ASS rescued test therapy is the generation of the helper-dependent animals from neonatal mortality and corrected adenoviral vector (HDV). In this vector, all viral coding hyperglutaminemia. There was also evidence of sequences are replaced by human genomic noncoding de novo urea-cycle activity, as measured in DNA [36,37]. Hence, there is no host-cell-mediated stable-isotope precursor-product studies [26,27]. immune response to HDV and no direct hepatotoxicity More recently, in a murine model of citrullinemia, an associated with viral gene expression. It has also been E1-deleted adenovirus expressing human ASS also suggested that modifying the tissue specificity of rescued the neonatal lethality, and corrected transgene expression might be important in biochemical parameters [28]. avoiding the immune clearance of the transgene However, in all the above models, ubiquitously product [38]. Although these features prevent the expressing promoters were used and only transient adaptive-immune-response-mediated phases of correction of the phenotype was achieved. Species adenoviral-vector toxicity, the HDV might not differences were also observed: 1013 vector particles/kg circumvent the immediate toxic effect on the processing transduced 100% of hepatocytes in mice but only of capsid proteins upon vector entry and/or direct 30% in the bovine model [26,27]. Furthermore, interaction with the innate immune system [35]. For cell-mediated immune responses against the vector some diseases, it might be possible to reduce this and/or the transgene protein are a major obstacle in acute toxicity by decreasing dose and increasing gene all animal models of adenoviral gene delivery [29], expression. A potential strategy for enhancing gene and these responses contribute to the chronic toxicity expression in viral vectors is to use the woodchuck- and transience of clinical correction that are usually hepatitis-virus post-transcriptional regulatory element observed in such models. In addition, acute toxicity (WPRE) [39]; inclusion of this element in the sense (e.g. direct hepatotoxicity associated with viral gene orientation downstream of the cDNA is postulated to expression, and transient thrombocytopenia) enhance mRNA stability [40]. Strategies aimed at further decreased the therapeutic index of these re-administration of adenoviral vectors have also vectors [30,31]. These studies also identified clear shown promise; antibodies against adenoviral vectors species differences in the post-translational are serotype-specific and, hence, the use of vectors with processing of UC enzymes. For example, newly different serotypes (e.g. type 5 versus type 2) enables synthesized mouse ornithine transcarbamylase re-administration [41]. Alternatively, modulating the (OTC) was efficiently imported into mouse immune response at the time of vector administration, mitochondria, following vector-mediated gene for example by inhibition of co-stimulatory molecules delivery in OTC-deficient mice, whereas human OTC important in B-cell antigen presentation, might was inefficiently imported [32]. This result might be prevent or reduce neutralizing antibody production. due to species-specific differences in the mitochondrial This approach was successfully used in a mouse model leader-peptide sequence of OTC [33]. of OTCD, in which co-administration of an anti-CD4 In a phase I and II human clinical study, an E1- and monoclonal antibody with the therapeutic vector, led E4-deleted adenoviral vector expressing human OTC to complete normalization of liver OTC activity and was administered into the right hepatic artery of metabolic correction [42]. http://tmm.trends.com 588 Review TRENDS in Molecular Medicine Vol.8 No.12 December 2002

Together, the modifications listed above should of vascular relaxation. Hence, any regulator of contribute significantly to increasing the therapeutic arginine availability might also affect NO synthesis. index of adenoviral gene therapy for cell-autonomous Support for this idea came from the recent discovery defects of hepatocyte metabolism, such as UCDs, and of a specific polymorphism (T1405N) within the CPS-I might allow this approach to be used for long-term gene, which was shown to be associated with an correction of these disorders in the future. Alternative increased frequency of neonatal pulmonary approaches, such as using adeno-associated virus hypertension and lower mean plasma arginine (AAV), are also being investigated. concentrations [48]. How hypomorphic or neomorphic variants of other enzymes in the UC affect arginine New UC-related genotype–phenotype correlations flux and, hence, NO production is still unclear. Recently, studies have linked alterations in the UC or UC-related processes with new disease pathologies Concluding remarks that present with hyperammonemia. For example, The UC is a unique pathway that is able to maintain a rare form of citrullinemia, known as ‘type II ammonia and urea homeostasis in the face of large citrullinemia’, has been described in Japan. This variations in nitrogen intake from dietary protein. disease usually presents in late childhood or in At the same time, it is able to compensate for adulthood, with a form of hepatocerebral disease peripheral mobilization of nitrogen during catabolic that is marked by hyperammonemia, elevated blood stress. The enzymatic components of the UC have citrulline, altered mental status and psychiatric been studied for many years, and this has led to a symptoms [43]. Type II citrullinemia results from a detailed understanding of the molecular pathway functional deficiency in liver ASS activity, even whose dysregulation results in UCDs. This has though ASS protein levels, mRNA and processing are facilitated the development of dietary and normal. It is caused by mutations in the SLC25A13 pharmacological therapies with underlying principles gene, which encodes a protein known as citrin [44], that are relevant to other inborn errors of hepatocyte thought to be a mitochondrial aspartate and metabolism. Traditional methods of diagnosis and glutamate carrier (Fig. 1). Pathogenesis is due to a management have relied on non-steady-state substrate deficiency of aspartate, which is required metabolite sampling, but recent advances have for citrulline incorporation into argininosuccinic shown that the direct in vivo measurement of acid (Fig. 1). The clinical phenotype has been metabolic flux might be a more precise diagnostic and recently expanded to include neonatal presentations prognostic indicator. However, the clinical application of hepatitis [43]. of this approach still faces problems. Another condition arising from a functional Owing to the significant morbidity and mortality UC deficiency is caused by mutations in a different associated with UCDs, cell therapy (i.e. orthotopic liver transporter molecule, SLC7A7 (Fig. 1). A lack of this transplantation) and gene-replacement therapy have dibasic-amino-acid transporter disrupts the reuptake been examined as potential methods for the long-term of ornithine, lysine and arginine, and results in correction of these disorders. Transplantation is lysinuric protein intolerance (LPI) [45]. Patients curative, but is associated with significant short-term with LPI excrete increased quantities of these amino and long-term morbidity, and gene-replacement acids in their urine. The resulting intracellular therapy, although extremely promising, is still a long ornithine deficiency in hepatocytes causes a way from being applied clinically. concomitant decrease in flux through the cycle and, Studies have shown that ureagenesis is regulated hence, hyperammonemia. both by proteins involved in the enzymatic conversion Finally, the condition ‘hyperornithinemia, of intermediary metabolites, and by transporters of hyperammonemia and homocitrullinuria’ these metabolites. Although much is known about the (HHH) is characterized by elevated levels of basic molecules required for ureagenesis, important ornithine and ammonia in the blood, and elevated questions remain: (1) can we adapt methods for Acknowledgements homocitrulline in the urine. The SLC25A15 measuring metabolic flux in UCDs and other inborn We thank O. Hernandez for administrative gene, which encodes a mitochondrial ornithine errors of metabolism for clinical practice?; (2) can assistance and S. Carter transporter, is mutated in this disorder (Fig. 1) [46]. further modifications in cell therapy and adenoviral for clinical assistance. We Hence, the pathogenesis of HHH, much like gene therapies be made, to increase their therapeutic are supported by the NIH that of LPI, is the result of an intracellular indices and make them more attractive for long-term (DK56787 and DK54450), the Baylor College of deficiency of ornithine, a required substrate of the correction?; and (3) do secondary defects of Medicine Child Health urea cycle. In the case of HHH specifically, this ureagenesis contribute to the pathogenesis of Research Center, the deficiency is intramitochondrial. other diseases (e.g. primary mitochondrial disease, Mental Retardation UC intermediates are essential not only for which results from the mitochondrial localization of Research Center (HD24064) and the Texas maintaining nitrogen homeostasis, but also in a OTC and CPS-I), or of more common disease Children’s Hospital crucial and related pathway, the nitric oxide processes related to NO function? By combining General Clinical Research (NO)–citrulline–arginine cycle [47]. Arginine is the genetic study of urea-cycle patients and Center (RR00188). This review is dedicated to the converted to citrulline by NO synthase (Fig. 1), and a urea-cycle-related disorders, we hope that these memory of Dr Peter Reeds. byproduct of this reaction is NO, a primary mediator questions will be answered.

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A. 97, 8021–8026 35 Roy-Chowdhury, J. and Horwitz, M.S. (2002) sector, discipline and geographic 17 Summar, M. (2001) Current strategies for the Evolution of adenoviruses as gene therapy region management of neonatal urea cycle disorders. vectors. Mol. Ther. 5, 340–344 J. Pediatr. 138 (Suppl. 1), S30–S39 36 Kochanek, S. (1999) High-capacity adenoviral Register for the free e-mail alerts 18 Burlina, A.B. et al. (2001) Long-term treatment vectors for gene transfer and somatic gene that updates you about the latest with sodium phenylbutyrate in ornithine therapy. Hum. Gene Ther. 10, 2451–2459 jobs in your field transcarbamylase-deficient patients. Mol. Genet. 37 Kochanek, S. et al. (2001) High-capacity ‘gutless’ Metab. 72, 351–355 adenoviral vectors. Curr. Opin. Mol. Ther. All positions previously available on 19 Maestri, N.E. et al. (1999) Neonatal onset 3, 454–463 BioMedNet’s Job Exchange are now ornithine transcarbamylase deficiency: 38 Pastore, L. et al. (1999) Use of a liver-specific available through a retrospective analysis. J. Pediatr. promoter reduces immune response to the http://www.sciencejobs.com 134, 268–272 transgene in adenoviral vectors. Hum. Gene Ther. 20 Maestri, N.E. et al. (1996) Long-term treatment of 10, 1773–1781 girls with ornithine transcarbamylase deficiency. 39 Glover, C.P. et al. (2002) Adenoviral-mediated, N. Engl. J. Med. 335, 855–859 high-level, cell-specific transgene expression:

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