Recombinant adenoviral vectors have many attractive features for gene therapy applications, including high transduction efficiencies in vivo and the ability to transduce non-dividing cells. However, the persistence of these vectors and their ability to be readministered are limited by the response of the host immune system to viral gene products, either the proteins of the viral capsid necessary for infection or viral proteins expressed in transduced cells. Dr. Eisensmith and his colleagues propose that safe, efficient and persistent adenovirus-mediated gene transfer may be achieved through vector modification, host immunomodulation, or the combined application thereof. Modification of adenoviral vectors to reduce or abolish late viral gene expression may increase the persistence of adenovirus-mediated gene transfer. To test this hypothesis, the investigators will construct novel E1/E4-deleted adenoviral vectors and adenoviral amplicon vectors in which most or all viral genes have been deleted. High titers of these latter vectors will be produced using a true adenoviral packaging cell line that does not rely on the use of helper virus. Host immunomodulation may also increase the persistence of adenovirus-mediated gene transfer. This hypothesis will be tested by developing and testing reagents designed to reduce the phagocytic, cytokine-releasing and antigen-presenting functions of macrophages, to limit T cell-mediated host immune responses by clonal deletion or anergization of T cells specific for adenoviral antigens or by inhibiting Th1 differentiation, or to specifically block the interaction between T cells and B cells that is necessary for the generation of a strong humoral immune response. Each of these approaches will be tested in vivo, first alone and then in combination if necessary, and if synergism is likely based on immunological studies, by expressing the mouse phenylalanine hydroxylase (PAH) gene in PAH-deficient Pahenu2 mice, an animal model of phenylketonuria. Because of the complete absence of humoral or cellular immune responses in Pahenu2 mice to the mouse PAH protein, this combination of transgene and animal model greatly simplifies the interpretation of experimental results regarding the immunogenicity of viral vectors administered in the presence or absence of various immunomodulatory strategies. Following treatment, serum phenylalanine (PHE) levels and hepatic levels of PAH and viral DNA will be monitored over time to determine efficacy and persistence. Histopathological and immunohistochemical studies will be performed to determine the mechanism of action of these procedures and their safety. Those approaches showing the most promise will be further tested to fully characterize the degree and duration of their immunosuppressive effects so that these may be minimized for potential future applications in humans. Strategies that are successful in the treatment of the phenylketonuria present in the Pahenu2 mouse should also be applicable to the treatment of may other human metabolic diseases for which there are not representative animal models.
| Krougliak, V A; Krougliak, N; Eisensmith, R C (2001) Stabilization of transgenes delivered by recombinant adenovirus vectors through extrachromosomal replication. J Gene Med 3:51-8 |
| Cheshenko, N; Krougliak, N; Eisensmith, R C et al. (2001) A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus. Gene Ther 8:846-54 |
| Kuzmin, A I; Galenko, O; Eisensmith, R C (2001) An immunomodulatory procedure that stabilizes transgene expression and permits readministration of E1-deleted adenovirus vectors. Mol Ther 3:293-301 |
