Adenoviruses as vectors for the delivery of foreign genes

Ads have been developed as vectors for the delivery of foreign antigens for immunization against other (non-Ad) infectious agents and for gene therapy. Ads are good vectors for the delivery of foreign DNA in that viral replication is not dependent upon host cell replication, they can be molecularly engineered to enter host cells and express foreign proteins without infectious viral progeny, and they are extremely safe immunogens that have already been administered to many people. For gene delivery, the E3 region, described above as a cluster of viral genes potentially controlling pathogenesis, has been physically or functionally deleted. Experience with Ad/SV40, described above, paved the way for these constructs by demonstrating that the Ad E3 region is not essential for viral replication. The E3 region can be replaced with foreign DNA: insertion of the hepatitis B surface antigen (HBsAg) gene in place of the partially deleted Ad E3 results in the expression of HBsAg which elicited an antibody response in hamsters. Recently, many additional foreign genes have been cloned into Ad vectors. Foreign genes have also been placed and expressed in other Ad sites including positions downstream from the Ad major late promoter (MLP). The Ad MLP is a very active transcriptional start site which results in the expression of large amounts of foreign proteins. To elicit higher titers against a given antigen, duplicate cloning of the gene into several serotypes is performed, permitting booster immunizations without augmentation of a serotype-specific anti-Ad-neutralizing antibody response. Since there has been extensive use of the Ad4 and 7 serotypes in young adults in the military, these types are obvious choices for such recombinant vaccines. The efficacy of Ad constructs designed for immunization is largely dependent upon viral replication and expression of foreign proteins.

Ad constructs designed for gene therapy are replication defective. Foreign genes are inserted into deleted El A and E1B regions. Growth of these vectors is dependent upon a cell line that is stably trans-fected with the E1A and E1B sequences. The viruses are infection competent and enter host cells where they efficiently transcribe inserted sequences without the production of significant progeny virus. Human trials have been attempted to test Ad constructs containing the cystic fibrosis gene, one of the numerous inserts placed into Ad vectors to date. Genes that can replace congenitally absent gene products and genes that can control cell growth or promote cell death of malignant cells have been engineered into Ads for therapeutic purposes. The p53 gene and the herpes simplex thymidine kinase gene (used with ganciclovir) are examples of this approach. Adenoviruses that have cell death genes such as Fas are also being used to control the overgrowth of vascular wall myocytes and endothelium after angioplasty. In many of these situations, there is a need for prolonged expression of the transgene or the requirement to re-administer the vector. However, the immune response to the initial injection of Ad, or even preexisting immunity from natural Ad infection, may preclude this possibility. A number of approaches to solve this problem have been undertaken in model systems, including short-term immunosuppression with FK506 or cyclosporine at the time of vector administration; oral or intrathymic tolerance with viral proteins; or using the immunosuppressive properties of overexpressed Ad E3. Inhibition of the antibody response to replication-deficient Ads containing overexpressed E3 genes has allowed readministration of an Ad vector containing a functional bilirubin conjugating enzyme for correction of an enzymatic defect in rat liver. Though the expression of Ad-inserted genes is prolonged in some tissues, many problems remain to be solved to ensure the success of Ad vectors as delivery systems for gene therapy; achieving long-term expression of foreign Ad-inserted genes in the desired tissue(s) and circumventing host immune responses that ultimately lead to rejection of delivered genes are significant issues that must be resolved. This very active area of research is likely to lead to significant advances in the near future.

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