Pre-clinical trials have demonstrated curative but transient correction of the coagulation defect in factor IX-deficient dogs using in vivo gene transfer with recombinant adenoviral vectors containing the canine FIX gene (Ad/cFIX). This transient expression results from a host immune response which eliminates the hepatocytes infected with virus. A humoral response also develops in these animals which interferes with secondary adenovirus transduction.
The aim of this proposal is to develop an adenoviral vector system capable of evading these host immune responses. The cDNA for three different immunoregulatory proteins will each be cloned into a recombinant adenovirus vector. The first is CTLA4Ig, a soluble antagonist of the CD28/B7 co-stimulatory pathway involved in both B and T cell activation. The second is the gp19k protein from the wildtype adenovirus E3 region which has been found to bind MHC I molecules in the endoplasmic reticulum and prevent its localization to the cell surface thus preventing T-cell activation. The last protein is the 14.7k protein also from the adenovirus E3 region, which has been found in mice to inhibit cell lysis by tumor necrosis factor. Ad/cFIX will be mixed with a recombinant adenoviruses containing the gene for one of the immunoregulatory proteins and used for hepatic gene transfer in mice. For each experiment, the plasma levels of cFIX, duration of expression, induction of a cytotoxic T-cell response, level of neutralizing antibodies, and function of native immune system will be evaluated. The most promising vector systems will be tested in the canine model.
