Measuring Ureagenesis In Vivo with Stable Isotopes Provides a Tool to Assess Efficacy of Gene Therapy for Ornithine Transcarbamylase Deficiency ♦ 727

OTCD is an excellent model to study in vivo gene therapy for inherited metabolic disorders: it is relatively frequent; it is a liver based enzyme deficiency and therefore readily accessible to gene transfer; the clinical outcome is poor using currently available therapy; and there are faithful animal models. We have previously reported (JBC 271:3639,1996) that an adenoviral vector containing the OTC cDNA is capable of metabolic correction in the OTCD sparse fur (Spf/Y) mice for 2-3 months. In considering a human gene therapy trial, a measure of efficacy requires long-term monitoring of changes in urea synthetic capacity following gene transfer. As OTC enzyme activity can only be measured directly by liver biopsy, an alternate non-invasive methodology is required. We have developed an orally administered stable isotope method, employing ¹⁵NH₄Cl which permits repeated measures of urea synthetic capacity (i.e. conversion of¹⁵ N ammonia into ¹⁵N urea and glutamine) in blood (JCI 98:2167,1996). To study the utility of this method, we administered intravenously 2×10¹¹ particles of an OTC expressing, E1 deleted adenoviral vector containing a temperature-sensitive mutation (ts125) in the E2 region to OTCD Spf/Y mice. ¹⁵N studies (5mmol/kg) were performed prior to and either 7 or 14 days following gene transfer. Mice were sacrificed at 7 or 14 days and liver OTC activity was measured. Aβ-galactosidase construct was used as a control; as expected, this construct had no effect on ¹⁵N incorporation into urea or on liver OTC activity in Spf/Y mice. The OTC containing construct, however, increased ¹⁵N incorporation into urea to 198±22% of pre-treatment levels on day 7 (n=6) and to 266±52% on day 14 (n = 6). Liver OTC enzyme activity increased to 91±10% of control (C3H mouse) activity on day 7 and 19±3% on day 14. These compare to 4±0.2% of control OTC activity in untreated Spf/Y mice. These results suggest that stable isotope studies provide a noninvasive and sensitive means of assessing outcome of gene therapy in OTCD over time. The study further shows that the increase in functional urea synthetic capacity exceeds changes in liver OTC activity. This suggests that even a rather small change in OTC activity induced by gene therapy may translate into a clinically significant improvement in urea synthetic activity and clinical symptoms.