Efficient mitochondrial import of newly synthesized ornithine transcarbamylase (OTC) and correction of secondary metabolic alterations in spf(ash) mice following gene therapy of OTC deficiency.

BACKGROUND: The mouse strain sparse fur with abnormal skin and hair (spf(ash)) is a model for the human ornithine transcarbamylase (OTC) deficiency, an X-linked inherited urea cycle disorder. The spf(ash) mouse carries a single base-pair mutation in the OTC gene that leads to the production of OTC enzyme at 10% of the normal level.

MATERIALS AND METHODS: Recombinant adenoviruses carrying either mouse (Ad.mOTC) or human (Ad.hOTC) OTC cDNA were injected intravenously into the spf(ash) mice. Expression of OTC enzyme precursor and its translocation to mitochondria in the vector-transduced hepatocytes were analyzed on an ultrastructural level. Liver OTC activity and mitochondrial OTC concentration were significantly increased (300% of normal) in mice treated with Ad.mOTC and were moderately increased in mice receiving Ad.hOTC (34% of normal). The concentration and subcellular location of OTC and associated enzymes were studied by electron microscope immunolocalization and quantitative morphometry.

RESULTS: Cytosolic OTC concentration remained unchanged in Ad.mOTC-injected mice but was significantly increased in mice receiving Ad.hOTC, suggesting a block of mitochondria translocation for the human OTC precursor. Mitochondrial ATPase subunit c [ATPase(c)] was significantly reduced and mitochondrial carbamy delta phosphate synthetase I (CPSI) was significantly elevated in spf(ash) mice relative to C3H. In Ad.mOTC-treated mice, the hepatic mitochondrial concentration of ATPase(c) was completely normalized and the CPSI concentration was partially corrected.

CONCLUSIONS: Taken together, we conclude that newly synthesized mouse OTC enzyme was efficiently imported into mitochondria following vector-mediated gene delivery in spf(ash) mice, correcting secondary metabolic alterations.