A deficiency of the urea cycle enzyme ornithine transcarbamylase (OTC) is associated with life-threatening episodes of hyperammonemia. This X- linked recessive disorder is an excellent model for developing liver- directed human gene therapies for several reasons. OTC deficiency (OTCD) is clearly a lethal disease with no effective therapy. Patients who experience hyperammonemia in the neonatal period have a 50% mortality with those survivors experiencing recurrent episodes of life.threatening hyperammonemia in childhood. Orthotopic liver transplantation has corrected the underlying defect in OTCD patients indicating that the hepatocyte in an appropriate target for gene therapy. Importantly, several authentic murine models of OTCD are available. We propose in this grant to develop approaches for treating OTCD using viral-based gene delivery systems. A major focus of the proposal is to develop recombinant adenoviruses for in vivo liver-directed gene therapy. Preliminary experience with this technology has confirmed its usefulness in achieving very efficient recombinant gene expression in liver. First generation recombinant adenoviruses, however, will have limited usefulness in treating OTCD because expression has been transient and associated with the development of inflammation. Our hypothesis to explain these important limitations is that the first generation adenoviruses express viral genomes which stimulate destructive CTL responses and the eventual repopulation of the liver with nontransgene containing cells. A major focus of this grant is to further define the immunologic basis for the transient gene expression and inflammation associated with the use of first generation adenoviruses. This will provide a rational basis for the development of second generation adenoviruses that express less viral proteins and, therefore, will confer more stable gene expression and less inflammation. This project will also develop the basic gene transfer technology necessary to perform ex vivo gene therapy in OTC-deficient patients to achieve partial, but stable, genetic reconstitution. Recombinant retroviruses will be designed, produced and tested in human primary cultures. In an attempt to further improve the effectiveness of ex vivo gene therapy, we will study the basic biology of retrovirus- mediated gene transfer in human hepatocytes and design methods for enhancing efficiency.
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