We propose to study novel approaches to the prolongation of recombinant adenovirus-mediated gene expression and the ability to readminister recombinant adenoviruses in rhesus monkey liver. These experiments are based on results in analogous studies in the mouse, as well as preliminary experiences in the rhesus. Interspecies variability seen in immune responses during gene transfer experiments suggests that non-human primates are a necessary study cohort prior to consideration of human clinical trials. Although modification of the adenoviral backbone to further limit late gene expression may prove fruitful, modulation of the host immune response is more likely to prove fruitful in the short term. The purpose of this grant is to describe experiments designed to explore the feasibility of transient, selective immunosuppression as a strategy to improve the usefulness of recombinant adenoviruses as gene therapy vectors by either prolonging transgene expression, or by prevention of neutralizing antibody which will allow readministration of the vector. The approaches will include transient immunosuppression of rhesus macaques with a series of immunosuppressive agents all of which have been selective specificity for their utility in preventing rejection in organ transplants, and to act at different points in the developing immune response. These regimens include: Single agent tacrolimus (FK506); Single agent OKT4A; Combination cyclophosphamide and corticosteroids; Combination CyA, 15-deoxyspergualin, and anti-thymocyte globulin (ATGam); and Combination cyclosporin A, corticosteroid, and mycophenolate mofetil. All of the proposed regimens, in addition to having been proved useful in primate transplantation, have been utilized clinically for human organ transplantation, and as such could be rapidly translated into human clinical trials if proven effective. Each regimen has been chosen for a different mechanism of action to selectively alter aspects of the host immune system. We will perform extensive characterization of the immune host response to recombinant adenoviruses including cytotoxic T lymphocyte assays, proliferation assays, cytokine release, neutralizing antibody assays, and Western blots. We will also study the duration of transgene expression and the ability to readminister recombinant adenoviruses. Lastly, toxicity will be analyzed by routine H and E histology. Preliminary studies with high-dose cyclophosphamide suggest profound lymphocyte depletion, and that a gnotobiotic environment-administered with the help of the proposed gnotobiology core- will be required to optimize safety for the monkeys.
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