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Oncolytic human adenovirus (Ad) vectors exert their antitumor effect by replicating in and lysing tumor cells. These vectors are commonly evaluated in immunodeficient mice bearing human tumor xenografts. However, this model suffers because the mice are immunodeficient and are not permissive for human Ads. We have developed a cotton rat model to test the selectivity, immunogenicity, and efficacy of oncolytic Ad vectors. The cotton rat is a rodent species that is semipermissive for human Ads. We show that the cotton cancer rat cell line LCRT supports the replication of human Ad in tissue culture and that the cells are destroyed on virus replication. When injected subcutaneously, LCRT cells formed tumors in immunocompetent cotton rats, and the growth of these tumors was delayed by the injection of an oncolytic Ad vector. Replication of the Ad vector in the tumor was demonstrated by sampling tumor tissue and isolating infectious virus particles at various times after intratumoral injection of the virus. We propose that the cotton rat can be used as an animal model to evaluate oncolytic Ad vectors.

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).