198. Tissue restricted overexpression of human ornithine transcarbamylase with a helper-dependent adenovirus containing the WPRE confers long-term correction in OTC-deficient mice

Document Type



Emergency Medicine


Urea cycle disorders have been models for development of human gene replacement therapy for cell autonomous disorders of hepatocyte metabolism. However, in preclinical studies in mouse models for ornithine transcarbamylase (OTC) deficiency, early generation adenoviral vectors expressing the mouse OTC gene, but not the human, achieved only transient correction (less than 8 weeks). This observation was later attributed to mitochondrial leader peptide sequence differences between the two species which decrease import of human OTC (hOTC), and to chronic toxicity associated with the early generation adenovirus. Moreover, the single human clinical trial failed to show significant clinical correction. We hypothesized that higher hOTC expression, from the less toxic helper-dependent adenoviral vector (HDV), would achieve long term correction in mice and serve as a preclinical model for the efficacy of such a vector in humans. To overexpress hOTC, we incorporated the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and the tissue-restricted phosphoenolpyruvate carboxykinase (PEPCK) promoter into the HDV hOTC vector. Intravenous administration of this vector restored liver OTC activity (as shown by biochemical and histochemical assays) in affected OTCspf-ash mice, and achieved long-term correction of orotic aciduria for at least 6 months (with measurements ongoing). An identical dose of HDV expressing hOTC, but without WPRE, was not efficacious. Southern analysis showed that the therapeutic efficacy of HDV PEPCK hOTC WPRE was not due to increased hepatocyte transduction by vector. Instead, we detected a 9 fold greater steady-state mRNA level, consistent with a posttranscriptional mechanism of enhanced gene expression. Protein levels were also elevated. Hence, by protein amplification, the WPRE was able to effectively increase the level of hOTC expression and overcome the kinetic block at the level of mitochondrial protein import. In conclusion, further development of HDVs using the WPRE for in vivo gene replacement for cell autonomous diseases such as urea cycle disorders is warranted. This vector-transgene combination exhibits decreased long-term toxicity, prolonged and greatly elevated transgene expression, and efficient hepatocyte transduction.


This work was published before the author joined Aga Khan University.

Publication (Name of Journal)

Molecular Therapy