Sustained correction of OTC deficiency in spf( ash) mice using optimized self-complementary AAV2/8 vectors.

Ornithine transcarbamylase deficiency (OTCD) is the most common inborn error of urea synthesis. Complete OTCD can result in hyperammonemic coma in the neonatal period, which can rapidly become fatal. Current acute therapy involves dialysis; chronic therapy involves the stimulation of alternate nitrogen clearance pathways; and the only curative approach is liver transplantation. Adeno-associated virus (AAV) vector-based gene therapy would add to current treatment options provided the vector delivers high level and stable transgene expression in liver without dose-limiting toxicity. In this study, we employed an AAV2/8-based self-complementary (sc) vector expressing the murine OTC (mOTC) gene under a liver-specific thyroxine-binding globulin promoter and examined the therapeutic effects in a mouse model of OTCD, the spf (ash) mouse. Seven days after a single intravenous injection of vector, treated mice showed complete normalization of urinary orotic acid, a measure of OTC activity. We further improved vector efficacy by incorporating a Kozak or Kozak-like sequence into mOTC complementary DNA, which increased the OTC activity by five or twofold and achieved sustained correction of orotic aciduria for up to 7 months. Our results demonstrate that vector optimizations can significantly improve the efficacy of gene therapy.

Alternative pathway therapy for urea cycle disorders: twenty years later.

Alternative pathway therapy is currently an accepted treatment approach for inborn errors of the urea cycle. This involves the long-term use of oral sodium phenylbutyrate, arginine supplements, or both, depending on the specific enzyme deficiency, and treatment of acute hyperammonemic crises with intravenous sodium benzoate/sodium phenylacetate plus arginine. A review of 20 years of experience with this approach illustrates the strengths and limitations of this treatment. It has clearly decreased the mortality and morbidity from these disorders, but they remain unacceptably high. The medications are generally well tolerated, but severe accidental overdosage has been reported because of the infrequent use of the medication. There is also a difference in their metabolism between newborns and older children that must be addressed in determining dosage. To avoid these complications it is recommended that drug levels in blood be monitored routinely and that very specific treatment protocols and oversight be followed to avoid overdoses. Finally, it must be acknowledged that alternative pathway therapy has limited effectiveness in preventing hyperammonemia and must be combined with effective dietary management. Therefore in children with neonatal-onset disease or in those with very poor metabolic control, liver transplantation should be considered. There should also be the continued search for innovative therapies that may offer a more permanent and complete correction, such as gene therapy.

Correction of argininosuccinate synthetase (AS) deficiency in a murine model of citrullinemia with recombinant adenovirus carrying human AS cDNA.

Citrullinemia is an autosomal recessive disorder caused by the deficiency of argininosuccinate synthetase (AS). It is characterized by elevated levels of blood citrulline and ammonia, which often results in hyperammonemic coma and early neonatal death in affected children. We have explored the use of adenoviral vectors as a treatment modality in a murine model of citrullinemia, the Ass mouse. The Ass mouse has no endogenous AS activity due to a targeted interruption of the AS gene. Homozygous mutant animals develop high levels of blood citrulline, become hyperammonemic, and die within 24-48 h after birth. We demonstrated that the neonatal crisis in Ass mice can be ameliorated by the injection of a recombinant adenovirus carrying human AS cDNA (Ad.CMVhAS) within hours after birth. The average life span of the virus-treated animals was extended from 30 +/- 9.5 h to 16.1 +/- 1.6 days. A second viral infusion 14 days after the first dose further prolonged the life span to an average of 36.2 +/- 7.0 days, and to 40.7 +/- 3.3 days with a concurrent daily injection of arginine and sodium benzoate. Significantly increased liver AS activity (47.3 +/- 7.9% of normal) was detected 24 h after viral infusion, which reached peak levels (80-90% of normal) at day 7 and decreased to about 20% of normal within 2-3 weeks after viral infusion. Southern blot analysis of liver DNA revealed a transduction efficiency of about one viral genome per hepatocyte 7 days after viral infusion and a gradual decrease of viral genome per cell parallel to the loss of liver AS activity. Plasma glutamine levels were partially normalized in virus-treated animals and were completely normalized in animals receiving Ad.CMVhAS concurrently with alternative pathway therapy. Plasma arginine levels were also partially normalized. Together, these results demonstrated that the recombinant adenovirus was capable of conferring AS activity in the liver of the recipient animals within 24 h, and the neonatal crisis of hyperammonemia could be averted by acute treatment with the AS containing adenovirus.

Treatment of urea cycle disorders.

Recent advances in the treatment of inborn errors of urea synthesis have significantly decreased mortality. Treatment has included combining a high-quality low-protein diet with supplements of deficient metabolites and stimulation of alternate pathways of waste nitrogen excretion. Long-term alternate pathway therapy, using sodium benzoate and sodium phenylacetate, has generally been unassociated with signs of toxicity. However, acute intoxications have simulated hyperammonemic crises. Neurologic outcome appears to be primarily a function of duration of neonatal hyperammonemic coma, although ongoing accumulation of urea cycle intermediates may also play a role. Early recognition and treatment are critical if a good outcome is to be possible.

Neurologic outcome in premature infants with transient asymptomatic hyperammonemia.

We studied the short-term and long-term effects of transient asymptomatic neonatal hyperammonemia on neurologic function in 21 preterm infants with normal ammonium levels and 25 with hyperammonemia (range 40 to 72 mumol/L) during the first weeks of life. The hyperammonemic infants were prospectively randomized to treatment with orally administered arginine free base 1 to 2 mmol/kg/day for 2 months (n = 13) or to a no-treatment control group (n = 12). Cortical function was assessed by auditory response and habituation during the first month of life. An auditory response was shown by 64% of the hyperammonemic infants and 43% of the normoammonemic infants (P not significant). Plasma ammonium levels at the time of examination bore no consistent relationship to whether an infant responded to an auditory stimulus. Number of trials to reach auditory habituation was also not different, and plasma ammonium level did not correlate with the presence or absence of habituation. IQ testing at 6, 12, 18, and 30 months showed no significant differences between groups. Early plasma ammonium levels did not have an effect on 30-month IQ scores. These findings suggest that transient asymptomatic hyperammonemia in premature infants is not associated with short-term or long-term neurologic deficits through 30 months of age. This study does not support the need for treatment of transient asymptomatic hyperammonemia in the premature infant.

Hyperammonemia.

A symptomatic elevation in plasma ammonium concentration, termed hyperammonemia, is associated with numerous congenital and acquired conditions (Table 11). In some cases, such as urea cycle disorders, ammonia is the principal toxin. In other instances, such as portal systemic encephalopathy, it is but one of a number of metabolic disturbances, However, in either case hyperammonemic episodes should be treated aggressively to prevent coma, subsequent brain damage, or death. This involves restricting protein intake, providing adequate calories, and giving agents that remove accumulated nitrogen. Long-term therapy relies on diagnosing the specific disease rate. This rarely requires invasive procedures such as liver biopsy. In most cases measurement of plasma amino acids and urinary organic acids will identify the defect. Treatment involving restriction of nitrogen intake, vitamin supplementation, or stimulation of alternative pathways of waste nitrogen excretion can then be instituted. Early therapy, especially in patients with neonatal-onset hyperammonemia, is imperative to avoid severe brain damage. On this basis, the plasma ammonium level should be determined in virtually every newborn with lethargy, hypotonia, poor feeding, seizures, and/or respiratory distress of unclear origin (Table 12).

Arginine-responsive asymptomatic hyperammonemia in the premature infant.

We found that more than 50% of premature infants have elevated plasma ammonium levels during the first 2 months of life. Ammonium levels were twice normal and were unaccompanied by clinical symptoms of vomiting or lethargy. Ten of these infants were given supplements of arginine (1 to 2 mmol/kg/day PO) for 1 to 2 weeks preceded and followed by control periods. In each infant, plasma ammonium levels fell significantly within 2 days of start of arginine supplementation, and increased once arginine was discontinued. We studied 59 additional premature infants, of whom 26 had normal ammonium levels and 33 were hyperammonemic. Plasma arginine and ornithine levels were significantly lower in the hyperammonemic group, but there was no difference in urinary excretion of arginine or ornithine between groups. Half of the hyperammonemic infants received arginine supplementation between 2 and 8 weeks of age. Plasma ammonium levels in the arginine group was 33 + 1 mumol/L., compared to 45 + 2 mumol/L in the untreated group. Follow-up at 18 months of age showed similar IQ scores in all groups, suggesting that significant neurologic deficits do not result from this transient metabolic defect. The mechanism of the hyperammonemia is unclear.

Treatment of inborn errors of urea synthesis: activation of alternative pathways of waste nitrogen synthesis and excretion.

Children with inborn errors of urea synthesis accumulate ammonium and other nitrogenous precursors of urea, leading to episodic coma and a high mortality rate. We used alternative pathways for the excretion of waste nitrogen as substitutes for the defective ureagenic pathways in 26 infants. These pathways involve synthesis and excretion of hippurate after sodium benzoate administration, and of citrulline and argininosuccinate after arginine supplementation. The children were treated for seven to 62 months; 22 survived. The mean plasma level of ammonium ( +/- S.E.) was 36 +/- 2 mumol per liter, and that of benzoate was 1.5 +/- 1.0 mg per deciliter. Alternative pathways accounted for between 28 and 59 per cent of the total "effective" excretion of waste nitrogen. Nineteen infants had normal height, weight, and head circumference, and 13 had normal intellectual development. Activation of alternative pathways of waste nitrogen excretion can prolong survival and improve clinical outcome in children with inborn errors of urea synthesis.

Treatment of hyperammonemic coma caused by inborn errors of urea synthesis.

The relative effectiveness of exchange transfusion, peritoneal dialysis, arginine, and sodium benzoate was evaluated during 44 episodes of hyperammonemic coma in 31 patients with congenital urea cycle enzymopathies. The overall survival rate was 56%. In 15 episodes treated with EXT the fall in ammonium was 19 +/- 24%, P > 0.05. In 30 episodes treated with PD, the fall in ammonium was 60 +/- 9%, P < 0.001. Ten times more nitrogen was removed as glutamine than as ammonium during dialysis, suggesting that the effectiveness of PD resides in the removal of glutamine, glutamate, and alanine as well as ammonium. Prior to therapy all patients had hypoargininemia (18 +/- 2 microM); they responded to arginine supplementation with a rise in plasma arginine concentration to normal. In patients with AL deficiency, arginine supplementation (4 mmol/kg/day) was associated with a fall in ammonium level from 917 +/- 62 to 103 +/- 18 microM within 24 hours. When sodium benzoate (250 mg/kg/day) was used during eight episodes of coma, six patients responded with a significant decrease in plasma ammonium.

Arginine therapy of argininosuccinase deficiency.

Argininosuccinic acid (A.S.A.) contains the two waste nitrogen atoms later excreted in urea in healthy people, and it has a renal clearance similar to the glomerular filtration-rate. Therefore, argininosuccinic acid might provide a vehicle for the excretion of waste nigrogen in patients with argininosuccinase deficiency, providing that stoichiometric amounts of ornithine are available. When two infants with no, or very little, erythrocyte argininosuccinase activity who were in neonatal hyperammonaemic coma were treated with supplementary arginine (4-5 mmol/kg/day) plasma ammonium, glutamine, and alanine concentrations became normal. One infant grew and developed normally during the first month of life on a protein intake of 2 g/kg/day. The other infant had sustained lathal brain damage before arginine therapy was tried and she died aged 17 days. Arginine supplementation may be effective therapy for argininosuccinase deficiency because it promotes A.S.A. synthesis and hence excretion of waste nitrogen. The effects of high plasma-A.S.A. concentrations are unknown.

Long-term management of a case of carbamyl phosphate synthetase deficiency using ketanalogues and hydroxyanalogues of essential amino acids.

A 13-year-old girl with congenital deficiency of carbamyl phosphate synthetase has been treated intermittently for one year with a restricted protein diet supplemented by various mixtures of the alpha-ketoanalogues of valine, leucine, isoleucine, and phenylalanine, the D,L-alpha-hydroxyanalogue of methionine, and five amino acids (lysine, arginine, histidine, threonine and tryptophan). Numerous adjustments in the composition of this mixture were made. Eventually normal levels of plasma ammonia and most amino acids were achieved, with three exceptions: slightly increased glutamine, pronounced alloisoleucinemia, and persistently low phenylalanine. Alloisoleucine was shown not to be incorporated into plasma protein and not to be excreted in the urine; hence this abnormality was viewed as being clinically insiginificant. Hypophenylalaninemia was unexplained, and failed to respond to increased phenylpyruvate dosage or phenylalanine itself; renal clearance of phenylalanine was high but could not account for the low plasma level. Compared to the pretreatment period her clinical status has improved markedly. Physical and mental development has continued at the same rate. Temporary withdrawal of the supplements led to prompt increases in plasma ammonia, glutamine, and alanine. We conclude that this therapy provides safe and effective long-term management for this patient's disorder and may be useful in other cases of congenital hyperammonemia.

Multidisciplinary management of dystonia misdiagnosed as hysteria.

Dystonia in the pediatric age group can be confused with hysteria, particularly when it occurs in an emotionally disturbed child with a negative family history of dystonia. A 20-year-old girl with a 12 year history of DMD is described. From age 12 to 17 she was housed in a mental institution after a misdiagnosis of hysteria was made. The progressive nature of DMD and the important emotional components are stressed. The multidisciplinary management model is discussed as a valuable method in the treatment of this chronic neurological disorder.