Gene Therapy of Ornithine Transcarbamylase Deficiency

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What is OTC deficiency ?

Ornithine transcarbamylase (OTC) deficiency is the most common urea cycle disorder with an incidence rate of 1:80,000 births in Japan (1). It occurs when a mutant enzyme protein (OTC) impairs the reaction that leads to condensation of carbamoyl phosphate and ornithine to form citrulline. This impairment leads to reduced ammonia incorporation, which, in turn, causes hyperammonemia. Ammonia is especially damaging to the nervous system, so ornithine transcarbamylase deficiency causes neurological problems as well as eventual damage to the liver.

The Otc gene coding for the OTC enzyme is mainly expressed in the liver and is intramitochondrial. The hepatic urea cycle is the major route for waste nitrogen disposal generated by protein and amino acid metabolism. Low-level synthesis of certain cycle intermediates in extrahepatic tissues [such as the kidney (2), and the intestine (3) makes a small contribution to waste nitrogen disposal.

In healthy individuals, ornithine arising in the cytosol is transported to the mitochondrial matrix where the OTC enzyme catalyses the condensation of ornithine with carbamoyl phosphate to produce the amino acid citrulline. The energy for the reaction is provided by the high energy anhydride of carbamoyl phosphate (Figure 1).

OTC Deficiency

Figure 1: Biosynthesis of citrulline.

Citrulline is made from ornithine and carbamoyl phosphate in one of the central reactions in the urea cycle. The produced citrulline is transported from the mitochondria to the cytosol where the remaining of the reactions of the urea cycle takes place.

Failure to incorporate carbamoyl phosphate into citrulline by condensation with ornithine results in an excess of both substrates for the reaction. The consequent increase in hepatic ornithine is often reflected in an elevated serum level. By contrast, excessive mitochondrial carbamoyl phosphate finds its way into the cytosol, where it functions as a substrate for the carbamoyl phosphate synthetase (CPS) II reaction, which occurs in the cytosol. This results in orotic acid, which is a normal intermediate in pyrimidine biosynthesis. Pyrimidine biosynthesis is regulated very tightly because it is a pathway involved in nucleic acid biosynthesis; thus, increases in urinary excretion of orotate are rarely observed in normal humans.

In humans the Otc gene is located on chromosomal location Xp21.1, which means that OTC affected females are usually carriers while males rarely survive past 72h of birth. Half of those survivors die in the first month, and half of the remaining by age 5. Prognosis is less clear in cases of adult onset OTC as detection of the disease is almost universally post symptomatic.


Like other urea cycle disorders, OTC affects the body's ability to get rid of ammonia, a toxic breakdown product of the body's use of protein. As a result, ammonia accumulates in the blood causing hyperammonemia. This ammonia travels to the various organs of the body.

Another symptom of OTC is a buildup of orotic acid in the blood. This is due to an anapleurosis that occurs with carbamoyl phosphate entering the pyrimidine synthesis pathway.

Ornithine transcarbamylase deficiency often becomes evident in the first few days of life, however it can present at middle age. An infant with ornithine transcarbamylase deficiency may be lacking in energy (lethargic) or unwilling to eat, and have poorly-controlled breathing rate or body temperature. Some babies with this disorder may experience seizures or unusual body movements, or go into a coma. Complications from ornithine transcarbamylase deficiency may include developmental delay and mental retardation. Progressive liver damage, skin lesions, and brittle hair may also be seen. Other symptoms include irrational behavior (caused by encephalitis), mood swings, and poor performance in school.

In some affected individuals, signs and symptoms of ornithine transcarbamylase may be less severe, and may not appear until later in life. Some female carriers become symptomatic later in life in times of metabolic stress. This can happen as a result of anorexia, starvation, malnutrition, pregnancy or even (in at least one case) as a result of gastric bypass surgery. It is also possible for symptoms to be exacerbated by extreme trauma of many sorts, including, (at least in one case) adolescent pregnancy coupled with severe stomach flu.


Current treatment includes a low-protein formula called keto-acid together with administration of sodium benzoate and/or sodium phenylbutyrate, both of which aim at bypassing the urea cycle and avoid the ammonia build-up in the body. Sodium benzoate conjugates with the amino acid glycine to form hippuric acid, which bypasses the urea cycle and is excreted in urine, whereas sodium phenylbutyrate is oxidized to phenylacetate, followed by conjugation with the amino acid glutamine and excretion in the urine bypassing the urea cycle as well. Some patients are also given the amino acid arginine as a supplement, which, for reasons that are unclear, causes more nitrogen to be excreted in the urine lowering blood ammonia.

In cases of OTC where enzyme production is low or non-existent, treatment consisting of low-protein diet and dietary supplementation are inadequate. In these cases, a liver transplantation is a treatment option.

Despite aggressive pharmacotherapy, patients are at high risk for repeated episodes of hyperammonemia and cumulative neurological morbidity and mortality. Hence, risk-benefit considerations make the development of clinical gene replacement therapy for OTC appropriate for long-term correction as well as for bridging therapy to alternative treatments such as liver transplantation.

OTC represents a perfect candidate for gene therapy for a number of reasons; the gene has been cloned; the disorder is relatively common; the current clinical outcome is poor; and there are authentic animal models. More specifically, two animal models of OTC deficiency, spf and spfash mice have been generated to simulate the disease.

Mouse models of OTC disease Spf and Spfash

The spf mouse carries a point mutation involving a C-to-A transversion in exon 4 of the OTC gene (4), and although OTC is produced in normal amounts, the mutation impairs enzymatic activity. The second mouse model, the spf with abnormal skin and hair (spfash), has a mutation that causes abnormal splicing of the OTC mRNA (5). Both strains express less than 10% residual OTC enzyme activity in liver and share the salient biochemical feature of human OTC deficiency including markedly increased urinary orotic acid concentration (4,5). The spfash mutation has a unique effect on OTC biogenesis. From the single mutant gene in males, two OTC precursors (pOTC) are produced, which together amount to only 10% of wild-type pOTC levels (5). One pOTC is normal in all characteristics of its structure and biogenesis except quantity and gives rise to the residual (5-10%) hepatic OTC activity in the spfash mice. The other pOTC is elongated, based on its apparent molecular weight. It is capable of uptake into mitochondria, where it is cleaved to an elongated mature subunit. It cannot assemble into an active trimeric structure, however, and appears to degrade rapidly within the mitochondrion without contributing to the detectable steady-state protein level or enzymatic activity.

Viral Gene Therapy of OTC

It has been suggested that based on OTC clinical evidence, as little as 3% normal enzyme activity is sufficient to ameliorate the severe neonatal phenotype, making OTC deficiency an ideal model for the development of liver-targeted gene therapy (15).

Several studies over the past decade have found the therapeutic effect of several different Adenoviral (Ad) vectors to be transient in the OTC deficient mouse models (6,7). The longest period of biochemical correction using an E1/E2-deleted Ad expressing the mouse OTC (mOTC) gene was 2 months (7). A relatively high dose (5x1011 total particles per mouse) of an adenovirus expressing human OTC (hOTC) could not correct the biochemical defect in adult OTC-deficient mice (7). A report by Ye et al. suggests that the differences between mouse and human mitochondrial leader peptide sequences of OTC may lead to inefficient import of hOTC into mouse mitochondria (8). An isolated report of short-term correction in spfash mice with Ad expressing hOTC at a relatively low dose of 5x1008 pfu/mouse has not been reproduced (9).

Morsy et al. showed that an Ad vector expressing hOTC was therapeutic in neonatal spfash mice; however, measured OTC activity in treated mice was significantly lower than that in wild-type littermates (10). This was attributed to a dominant negative effect of the endogenous mutant protein on the activity of the wild-type protein delivered by the Ad. Morsy et al. also suggested that increased enzyme activity in the intestine at early time points may have contributed to the rapid reduction of urinary orotic acid (10) and this was consistent with earlier transgenic studies documenting the importance of small-intestine expression of OTC.

Increasing gene expression offers another potential avenue for increasing the therapeutic index, especially for a non-cell-autonomous process that would allow for an associated reduction in vector dose. The ability of specific viruses to enhance gene expression post-transcriptionally could serve well in this arena [11,12]. For example, Loeb et al. have postulated that the inclusion of the WPRE element in the sense orientation downstream of the cDNAs will enhance stability of mRNA [12]. Hence, it may be an effective tool for increasing the level of protein expression in gene therapy, as shown by Mian et al (10) in a recent report utilising a WPRE-mediated over expression of OTC using a Helper-Dependent Adenovirus.

When gene therapists utilised an alternative viral vector (Adeno-Associated Vector- AAV) for delivery of the Otc gene, a marked improvement in gene expression was observed. For example Moschioni et al, reported long-term correction of ammonia metabolism and prolonged survival of spfash mice by using an AAV vector delivering the cDNA of mOTC gene (13).

In another report, Cunningham et al. investigated the metabolic correction in neonatal and adult male OTC-deficient spfash mice following adeno-associated virus (AAV)2/8-mediated delivery of the murine OTC cDNA under the transcriptional control of a liver-specific promoter (15). Substantially supraphysiological levels of OTC enzymatic activity were readily achieved in both adult and neonatal mice following a single intraperitoneal (i.p.) injection, with metabolic correction in adults being robust and life-long. In the neonates, however, full metabolic correction was transient, although modest levels of OTC expression persisted into adulthood.  This loss of expression in the neonatal liver is the consequence of hepatocellular proliferation and presents an added challenge to human therapy.

The gene therapy research community was rocked by the death of Jesse Gelsinger, a volunteer in a phase I study where a gene for ornithine transcarbamylase was introduced using an adenoviral vector. This adenoviral vector caused a massive immune reaction causing death from multiple organ failure four days later.

This unfortunate example clearly demonstrated the necessity for safer alternative vectors such as plasmid DNA vectors, which could provide non-toxic long-term therapy.

Non-Viral Treatment:

Sadly, so far there has been no report of a non-viral vector for the treatment of OTC disease.

Clinical trials of OTC deficiency

A search in reveals 5 studies concerning OTC deficiency management in humans. Only one is currently active  (NCT00004498) using and Adenovirus vector, whereas two studies are focusing on the metabolic management of OTC.

1. (NCT00004498), Active, not recruiting:

Phase I Study of Adenoviral Vector Mediated Gene Transfer for Ornithine Transcarbamylase in Adults With Partial Ornithine Transcarbamylase Deficiency

2. (NCT00004386), Terminated Phase I:

Pilot Study of Liver-Directed Gene Therapy for Partial Ornithine Transcarbamylase Deficiency

3. (NCT00004307), Recruiting:

Study of Treatment and Metabolism in Patients With Urea Cycle Disorders

4. (NCT00472732), Recruiting:

Neurologic Injuries in Adults With Urea Cycle Disorders.

5. (NCT00718627), Recruiting:

Human Heterologous Liver Cells for Infusion in Newborns and Infants With Urea Cycle Disorders

Treating Ornithine TransCarbamylase (OTC) genetic disorder using plasmid DNA vectors – Our group efforts

As mentioned before, there have been no reported studies using a plasmid DNA (pDNA) vector for OTC gene therapy. Therefore, our group strategy was to combine a pDNA vector linked to a Scaffold Matrix Attachment Region (S/MAR) element, which has been shown before to confer long-term transgene expression in the mouse liver (14). The novel S/MAR vector (called pLucA1-OTC-WPRE), which codes for the human OTC cDNA followed by the WPRE element, was then administered using hydrodynamic delivery in adult male spfash mice.

Histochemical detection of hOTC in the mouse liver

An initial histochemical stain for OTC activity was used to characterize the distribution and level of OTC expression (Figure 2). This assay has been described before (7) and is based on the demonstration of a dark brown reaction product in the OTC expressing cells. In an untreated spfash homozygote mouse, very small to none areas of the liver are stained black, whereas a wild type mouse (such as an MF1 mouse) shows a 100% black staining of cells revealing normal OTC protein production.

Our histochemical analyses of liver sections isolated from spfash mice at 24h and one week following pDNA hydrodynamic delivery, clearly illustrate hOTC histochemically (Figure 2), As can be seen in figure 2 C-D for pLucA1-OTC-WPRE control (without S/MAR), and E-F for pLucA1-OTC-WPRE vector, a semiquantitative estimate of approximately 30-40% of hepatocytes within sections of treated mice appeared to be transfected compared to sections prepared from normal wild-type mice. OTC expression appears to be concentrated primarily in hepatocytes surrounding central and portal veins, a finding that agrees well with the expression profile of hepatocytes after hydrodynamic delivery seen before (10).

Expression of OTC enzyme in spfash strains treated with pLucA1-OTC-WPRE control vector, did not last beyond 24h as seen in figure 2 (D), whereas mice injected with pLucA1-OTC-WPRE pDNA were showing a sustained OTC expression figure 2 (F).

OTC Gene Therapy

Figure 2: Histochemical analysis of spfash mice injected with the pDNA, 24h and one week post injection.

Representative micrographs of liver sections taken at 24h and one week following hydrodynamic injection of pLucA1-OTC-WPRE and pLucA1-OTC-WPRE control vectors conferring the Otc gene. (A) MF1 mouse liver; (B) untreated spfash mouse; (C) pLucA1-OTC-WPRE control treated liver, 24h after injection; and (D) same vector one week post injection; (E) spfash mouse liver treated with pLucA1-OTC-WPRE, 24h and (F) one week post administration. Magnification x10.


There has been significant progress over the last years in developing novel vectors for the genetic therapy of Ornithine Transcarbamylase Deficiency. The vast majority of these vectors are of viral origin, but non-viral vectors have also started being investigated as a safer alternative. Further progress in delivery techniques, and prolonged gene expression will provide a better clinical treatment and outcome of OTC patients.


1) Estimated frequency of urea cycle enzymopathies in Japan. Nagata N, Matsuda I, Oyanagi K. Am J Med Genet. 1991 May1;39 (2):228-9

2) Purification and properties of arginase of rat kidney Kaysen, G.A. and H.J. Strecker. Journal of Biochemisrty,1973. 133: p. 779-788.

3) Developing adenoviral-mediated in vivo gene therapy for ornithine transcarbamylase deficiency Raper S.E., Wilson JM, Yudkoff M, Robinson MB, Ye X, Batshaw ML.  J Inherit Metab Dis, 1998. 21 Suppl 1: p.119-37.

4) The molecular basis of the sparse fur mouse mutation Veres, G. Gibbs, RA, Scherer SE, Caskey CT. Science,1987. 237(4813): p. 415-7.

5) Biogenesis of ornithine transcarbamylase in spfash mutant mice: two cytoplasmic precursors, one mitochondrial enzyme Rosenberg, L.E., F. Kalousek, and M.D. Orsulak, Science,1983. 222(4622): p. 426-8.

6) Patient selection may affect gene therapy success. Dominantnegative effects observed for ornithine transcarbamylase in mouse and human hepatocytes.Morsy, M.A. Zhao JZ, Ngo TT, Warman AW, O'Brien WE, Graham FL, Caskey CT Journal of  Clinical Investigation, 1996. 97: p. 826 – 832.

7) Prolonged metabolic correction in adult ornithine transcarbamylase-deficient mice with adenoviral vectors Ye, X., Robinson MB, Batshaw M, Furth EE, Smith I, Wilson JM. J Biol Chem, 1996. 271(7): p. 3639-46.

8) Differences in the human and mouse amino-terminal leader peptides of ornithine transcarbamylase affect mitochondrial import and efficacy of adenoviral vectors Ye, X., Zimmer KP, Brown R, Pabin C, Batshaw M, Wilson JM, Robinson MB. Human Gene Therapy, 2001. 12: p. 1035-1046.

9) Correction of ornithine transcarbamylase deficiency in adult spf(ash) mice and in OTC-deficient human hepatocytes with recombinant adenoviruses bearing the CAG promoter Kiwaki, K, Kanegae Y, Sito I, Komaki S, Nakamura K, Miyazaki JI, Endo F, Matsuda I. Human Gene Therapy, 1996. 7(7): p.821-830.

10) Long-term correction of ornithine transcarbamylase deficiency by WPRE-mediated overexpression using a helper-dependent adenovirus Mian, A, McCormack WM Jr, Mane V, Kleppe S, Ng P, Finegold M, O’Brien WE, Rodgers JR, Beaudet AL, Lee B. Mol Ther 2004. 10(3): p. 492-9.

11) Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element Donello, J.E., J.E. Loeb, and T.J. Hope, J Virol, 1998. 72(6): p. 5085-92.

12) Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy. Loeb, J.E, Cordier WS, Harris ME, Weitzman MD, Hope TJ, Human Gene Therapy, 1999. 10(14): p. 2295-305.

13) Long-term correction of ammonia metabolism and prolonged survival in ornithine transcarbamylase-deficient mice following liver-directed treatment with adeno-associated viral vectors. Moscioni D, Morizono H, McCarter RJ, Stern A, Cabrera-Luque J, Hoang A, Sanmiguel J, Wu D, Bell P, Gao GP, Raper SE, Wilson JM, Batshaw ML. Mol Ther. 2006 Jul;14(1):25-33.

14) Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector. Argyros O, Wong SP, Niceta M, Waddington SN, Howe SJ, Coutelle C, Miller AD, Harbottle RP. Gene Ther. 2008 Dec;15(24):1593-605.

15) AAV2/8-mediated correction of OTC deficiency is robust in adult but not neonatal Spf(ash) mice. Cunningham SC, Spinoulas A, Carpenter KH, Wilcken B, Kuchel PW, Alexander IE. Mol Ther. 2009 Aug;17(8):1340-6.