Replicationdefective Adenovectors

Most replication-defective adenovectors are generated by replacing the E1 region with a therapeutic gene (Fig. 1). Up to 3.2 kb of an E1 region can be removed. Adenoviruses can effectively package 105% of the length of the wild-type gene genome (37,38). Removal of the E1 region provides a total of packaging capacity about 5 kb for foreign inserts (39). Removal of the E3 region may provide another 2.7 kb of packaging space without producing dramatic effects on replication and viral stability (38,39). Although the genes in the E3 region are dispensable for the viral life cycle, those in the E1 region function as transactivators for other viral gene expression and are required for viral replication. Removal of the E1 region renders a virus defective in lytic replication. Therefore, whereas a vector can express the genes it carries, it cannot replicate in most cells. Nevertheless, vectors can be generated and propagated in packaging cells, such as 293 and 911 cells, those which provide E1 region functions in trans (40,41). Several methods have been reported for generating such vectors, including homologous recombination in packaging cells (42), homologous recombination in bacteria (43), and in vitro ligation (44,45).

Various in vivo studies have demonstrated that an E1-deleted adenovector can efficiently deliver transgenes to a variety of tissues in various animal models, including mouse, rat, rabbit, swine, dog, and monkey. However, these studies have also revealed that the transgene expression and therapeutic effects that occur in adenovirus-mediated gene transfer in immunocompetent animals are transient, primarily because of the host immune response to the E1-deleted recombinant adenoviral vectors and to the transgene product expressed in transduced cells (46-48). This transient expression suggests that repeated administration would be required for treating most genetic disorders and cancers. Unfortunately, in these studies host humoral immune responses to the vectors abrogated any therapeutic effects of the adenovirus-mediated gene transfer after repeated systemic administration (47,49).

Because of this problem, numerous efforts have been made to modify adenovectors by removing or inactivating other viral genes, including those in the E2 (50) and E4 regions (51-53) and in most of the entire viral coding regions (54-56). A packaging cell line for adenovectors in which most of an encoding region or the entire region has been removed is not available. Adenovectors with such large deletions are produced using a helper virus that contains all the viral genes required for replication but that has a conditional defect in its packaging (54,56). In immunocompetent animals, these modified vectors have been shown to be less toxic and have less immunogenicity and mediated prolonged transgene expression (57,58).

Additional strategies for improving adenoviral vector-based gene delivery systems have also been explored. For example, chemically or genetically modified capsid proteins to enhance the transduction efficiency in CAR-defective cells or to redirect vector tropisms have been rigorously tested and it has been reported that the formulation of an adenovector in protamine or other pharmaceutical excipients enhance in vivo transduc-tion in the lung after systemic administration, intratracheal instillation, or aerosolized vector delivery (59,60). Furthermore, several small molecules, such as Syn3 and some anticancer agents (61,62), have been reported to dramatically enhance adenovector-mediated gene delivery. Treating animals with low-dose etopside can suppress the formation of neutralizing antibodies and thus enhance intratumoral transgene expression in immunized animals (63). Moreover, the modification of adenovectors with polyethylene glycol (64-68) and formulations of adenoviral vectors in a collagen-based matrix (69), in a liposome, and in a synthetic surfactant (70-72) have all been reported to improve gene transfer in animals with antiadenovirus immunity. Several clinical trials have also demonstrated favorable outcomes in patients treated with adenovector-mediated cancer gene therapy plus conventional chemotherapy or radiation therapy. Although the mechanisms are yet to be determined, gene delivery by such a combination regimen has been shown to be enhanced in vitro in a cultured cell system.

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