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 o f argininosuccinate synthetase (AS). It is characterized by elevated levels of blood citrulline and ammonia, which often results in hyperammonemic com a and early neonatal death in affected children. We have explored the use o f adenoviral vectors as a treatment modality in a murine model of citrullin emia, the Ass mouse. The Ass mouse has no endogenous AS activity due to a t argeted interruption of the AS gene. Homozygous mutant animals develop high levels of blood citrulline, become hyperammonemic, and die within 24-48 ho urs after birth. We demonstrated that the neonatal crisis in Ass mice can b e ameliorated by the injection of a recombinant adenovirus carrying human A S cDNA (Ad.CMVhAS) within hours after birth. The average life span of the v irus-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 l ife 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 Significantly increased l iver AS activity (47.3+/- 7.9% Of normal) was defected 24h after viral infu sion, which reached peak levels (80-90% of normal) at day 7 and decreased t o 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 o f viral genome per cell parallel to the loss of liver AS activity. Plasma g lutamine levels were partially normalized in virus-treated animals and were completely normalized in animals receiving Ad.CMVhAS concurrently with alt ernative pathway therapy. Plasma arginine levels were also partially normal ized. Together these results demonstrated that the recombinant adenovirus w as 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. benzoate.