Adenovirus Mediated Gene Therapy in Citrullinemia: Clinical Correlations of in vivo Nitrogen Flux Measurements in Neontal Bovine Citrullinemia and in Urea Cycle Patients • 723

Despite progress in dietary and pharmacological therapy, the prognosis for urea cycle disorder patients remains poor and gene therapeutic interventions have been proposed. Precise measurements of urea cycle-specific nitrogen flux and ureagenesis in patients before and after such treatment will be a critical and sensitive tool for evaluating the efficacy of future gene therapy trials and in the diagnosis and clinical management of these disorders. We have determined the in vivo flux through the urea cycle pathway by measuring the conversion of intravenously infused [¹⁵N-amide]glutamine to [¹⁵N]urea. Measurement of total and endogeneous urea flux varied with protein intake and drug administration. An index of the proportion of urea synthesis from glutamine as measured by[¹⁵N]urea/[¹⁵N]glutamine correlated with severity of clinical phenotype and differentiated neonatal onset conditions characterized by absence of enzyme activity, from late onset OTC males and heterozygous females with partial activity, and from normal adult controls. This index in combination with absolute measurements of urea synthetic rates will be useful for evaluating gene therapeutic interventions and for guiding the clinical management of urea cycle patients.

As part of these preclinical studies, we have also intravenously administered a first generation adenovirus vector with a CAG promoter/argininosuccinate synthetase (AS) cDNA cassette into a neonatal calf model of citrullinemia. Measurements of blood chemistries and hematological indices revealed no adverse effects. Stable isotope studies with orally administered [¹⁵N]NH₄Cl before and after treatment revealed significant enrichment of [¹⁵N]urea consistent with newly transduced AS activity. This correlated with improvement in serum glutamine and ammonia levels, and clinical status of the affected calves. The large animal bovine model and the sensitivity of the stable isotope method will allow for the evaluation of efficacy and safety of adenoviral vectors in phase I/II clinical trials involving gene therapy of citrullinemia and other urea cycle disorders.