Characterization of an Adenovirus Gene Transfer Vector Containing an E4 Deletion

Abstract

We describe the construction and characterization of an adenovirus type 2 vector, Ad2E4ORF6, which has been modified in the E4 region to contain only open reading frame 6. When assayed in cultured cells, Ad2E4ORF6 virus replication is slightly delayed but viral DNA synthesis, host-cell protein synthesis shut-off, and virus yield are indistinguishable from wild type. Late protein synthesis is normal with the exception of fiber synthesis, which is reduced approximately 10-fold. Despite the reduced fiber synthesis, Ad2E4ORF6 viral particles appear to contain a full complement of fiber protein. Virus replication in cotton rats indicates that Ad2E4ORF6 is replication defective in vivo. This may have safety implications for the development adenovirus vectors in that virus arising by recombination in the E1 region of an Ad2E4ORF6-based vector would be defective for growth in vivo. The deletion of E4 open reading frames that are not required for virus growth in vitro increases the cloning capacity of adenovirus vectors by 1.9 kb and may be generally useful for the construction of adenovirus vectors containing large cDNA inserts and/or regulatory elements. We describe the inclusion of the A2E4ORF6 modification in a recombinant adenovirus vector, Ad2/CFTR-2, for gene transfer of the human cystic fibrosis transmembrane regulator (CFTR). Adenoviruses are increasingly being utilized as vectors for efficient gene transfer. Simple E1 replacement vectors that retain E3 have a cloning capacity of approximately 5 kb. To accommodate large cDNA inserts or expression cassettes, we chose to increase vector cloning capacity by deleting open reading frames of E4 that are nonessential for virus growth in cultured cells. A new adenovirus vector, Ad2E4ORF6, has been modified in the E4 region to contain only open reading frame 6. Although this vector is replication competent in vitro, it is replication defective in vivo. Incorporation of this partial E4 deletion into a new vector for cystic fibrosis gene therapy, Ad2/CFTR-2, has alleviated the poor growth characteristics observed with a previously described vector, Ad2/CFTR-1.