The biosynthesis of urea from ammonia and aspartate occurs predominantly in the liver through a series of four enzymes that form a biochemical cycle called the urea cycle. Each enzyme in this pathway is associated with an inherited deficiency state in humans that is characterized by hyperammonemia which is often life-threatening. The most common inborn error of urea metabolism is a deficiency of the ornithine transcarbamylase (OTC) enzyme. This x-linked recessive disorder is associated with variable hyperammonemia and pronounced orotic aciduria. This proposal describes new approaches to the treatment of OTC deficiency which are based on liver-directed gene transfer of a functional OTC gene. OTC deficiency has several features which make it an attractive candidate for liver-directed gene therapy: (1) The normal OTC gene has been cloned and is available; (2) Metabolic correction will require expression of normal OTC in hepatic mitochondria, suggesting that gene transfer must be targeted to hepatocytes as opposed to other somatic tissues such as bone marrow or skin; (3) Two authentic murine models of OTC deficiency have been described; (4) In its most sever form, OTC deficiency in humans is a life-threatening inherited disease that is resistant to conventional therapies. The murine animal models of OTC deficiency will be used to develop gene replacement therapies based on the delivery of a functional OTC gene to hepatocytes in vivo. Proposed strategies for targeted gene delivery to hepatocytes in vivo are based on interactions of the DNA/protein complexes with the hepatocyte-specific receptor, the asialogylcoprotein receptor. This in vivo transfection system will be used to identify transcriptional elements which confer high level expression of a transferred OTC gene. Stable expression of a transferred gene will be achieved through the development of vector systems that either integrate into genomic DNA or persist as extra chromosomal elements. The development of liver-directed gene therapies in murine models of OTC deficiency should greatly facilitate the eventual application of these technologies to the treatment of patients with OTC deficiency or other liver-specific disorders.
