AAV2/8-mediated Correction of OTC Deficiency Is Robust in Adult but Not Neonatal Spfash Mice

Abstract

Ornithine transcarbamylase (OTC) deficiency, the most common urea cycle disorder, is associated with severe hyperammonemia accompanied by a high risk of neurological damage and death in patients presenting with the neonatal-onset form. Contemporary therapies, including liver transplantation, remain inadequate with considerable morbidity, justifying vigorous investigation of alternate therapies. Clinical evidence suggests that as little as 3% normal enzyme activity is sufficient to ameliorate the severe neonatal phenotype, making OTC deficiency an ideal model for the development of liver-targeted gene therapy. In this study, we investigated metabolic correction in neonatal and adult male OTC-deficient Spf(ash) mice following adeno-associated virus (AAV)2/8-mediated delivery of the murine OTC complementary DNA under the transcriptional control of a liver-specific promoter. Substantially supraphysiological levels of OTC enzymatic activity were readily achieved in both adult and neonatal mice following a single intraperitoneal (i.p.) injection, with metabolic correction in adults being robust and life-long. In the neonates, however, full metabolic correction was transient, although modest levels of OTC expression persisted into adulthood. Although not directly testable in Spf(ash) mice, these levels were theoretically sufficient to prevent hyperammonemia in a null phenotype. This loss of expression in the neonatal liver is the consequence of hepatocellular proliferation and presents an added challenge to human therapy.

In vivo assessment of mutations in OTC for dominant-negative effects following rAAV2/8-mediated gene delivery to the mouse liver

“…Importantly, this confirmed that the vectors encoded stable OTC proteins that were successfully targeted to the liver. Consistent with our earlier findings, 6 the mutant OTC expression levels achieved were markedly in excess of endogenous physiological wild-type OTC levels.…”

“…The level of urinary orotic acid in affected adult mice is significantly higher at 629 ± 70 mmol orotate per mmol creatinine (n ¼ 8). 6 This implies that these mutant proteins do not exert a phenotypically significant dominant-negative effect on wild-type OTC.…”

“…5 In the spf ash mouse model of OTC deficiency, our group has shown liver-wide murine OTC expression after rAAV2/8-mediated delivery. 6 At a vector dose of 1.5 Â 10 12 vector genomes (vg) per mouse, OTC enzyme activities over an order of magnitude above physiological levels were observed, with complete biochemical correction achieved at a substantially lower dose of 1.5 Â 10 11 vg per mouse.…”

“…injection of 1 Â 10 12 vg per mouse, a dose shown earlier by our group to produce liver-wide gene transfer 12 and induction of super-physiological OTC protein levels. 6 Base-line and weekly urinary orotic acid levels were determined and compared with values obtained for control mice that received either wild-type OTC-encoding vector or phosphate buffered saline (PBS). Six weeks later the mice were killed and the livers harvested.…”

“…The DNA sequences of mutant OTC genes were confirmed to ensure that no spurious mutations had been introduced. Mutant OTC genes were sub-cloned into the Eco RI and Eco RV restriction endonuclease sites of pLSP1-mOTC 6 to replace the wild-type transgene. Figure 2 Level of urinary orotic acid after liver-directed gene delivery.…”