Hepatocyte gene therapy in a large animal: a neonatal bovine model of citrullinemia

Abstract

The development of gene-replacement therapy for inborn errors of metabolism has been hindered by the limited number of suitable large-animal models of these diseases and by inadequate methods of assessing the efficacy of treatment. Such methods should provide sensitive detection of expression in vivo and should be unaffected by concurrent pharmacologic and dietary regimens. We present the results of studies in a neonatal bovine model of citrullinemia, an inborn error of urea-cycle metabolism characterized by deficiency of argininosuccinate synthetase and consequent life-threatening hyperammonemia. Measurements of the flux of nitrogen from orally administered 15NH4 to [15N]urea were used to determine urea-cycle activity in vivo. In control animals, these isotopic measurements proved to be unaffected by pharmacologic treatments. Systemic administration of a first-generation E1-deleted adenoviral vector expressing human argininosuccinate synthetase resulted in transduction of hepatocytes and partial correction of the enzyme defect. The isotopic method showed significant restoration of urea synthesis. Moreover, the calves showed clinical improvement and normalization of plasma glutamine levels after treatment. The results show the clinical efficacy of treating a large-animal model of an inborn error of hepatocyte metabolism in conjunction with a method for sensitively measuring correction in vivo. These studies will be applicable to human trials of the treatment of this disorder and other related urea-cycle disorders.

Figure 1

Schematics of stable-isotope studies. (A) This 2-day stable-isotope study (represented by ovals in B) consists of ¹⁵NH₄Cl feedings every 6 h, preceded by blood sampling. Metabolic parameters were measured every 6 h and are listed under the time line. (B) Time line (in days) of citrullinemic-calf studies (S2 and S3) and control-calf studies (S6 and S7). The durations of arginine and/or sodium benzoate treatments are shown above the time lines. Ovals represent 2-day stable-isotope studies. For citrullinemic calves (S2 and S3), the days are listed relative to the day of virus infusion (day 0). For control calves (S6 and S7), the days are listed relative to the start of pharmacotherapy with benzoate and arginine (day 0).

Figure 2

The efficiency of adenoviral transduction after i.v. delivery. Staining for β-galactosidase expression in the right lobe of the liver (A), the left lobe of the liver (B), the spleen (C), and the lungs (D) shows only transduction of hepatocytes (×100).

Figure 3

Blood [¹⁵N]urea enrichment in control calves and the effects of arginine (Arg) and/or sodium benzoate (Benz) therapy (Rx). (A) Absolute [¹⁵N]urea enrichment (mol% excess) shown as time course of each 2-day stable-isotope study. Measurements were taken at 6-h intervals before feedings with milk and ¹⁵NH₄Cl. Studies without medications (♦), with arginine and sodium benzoate (■), and with only arginine (▴) show that steady-state levels are achieved after three to five doses of ¹⁵NH₄Cl. (B) Average [¹⁵N]urea enrichments over each 2-day isotope study from S6 and S7 on various therapeutic regimens. Determinations were made from blood samples obtained after the third dose of ¹⁵NH₄Cl at steady state. Error bars represent SD.

Figure 4

Efficacy of treatment of citrullinemic calves with AdΔE1CAGASS. (A) Average [¹⁵N]urea enrichment in S2 and S3 before vector treatment and at time points after vector treatments (error bars represent SD). The posttreatment (days 6 and 7) study on calf S3 was not done (ND), because the calf experienced gastroenteritis. (B) Time course of plasma glutamine levels in an untreated citrullinemic calf (▴) and in treated S2 (♦) and S3 (■) citrullinemic calves (ASS −/−). Control normal calves have average plasma glutamine levels as shown.