Despite recent technical improvements, the construction of recombinant adenovirus vectors remains a time-consuming procedure which requires extensive manipulations of the viral genome in both Escherichia coli and eukaryotic cells. This report describes a novel system based on the cloning and manipulation of the full-length adenovirus genome as a stable plasmid in E. coli, by using the bacterial homologous recombination machinery. The efficiency and flexibility of the method are illustrated by the cloning of the wild-type adenovirus type 5 genome, the insertion of a constitutive promoter upstream from the E3 region, the replacement of the E1 region by an exogenous expression cassette, and the deletion of the E1 region. All recombinant viral DNAs were shown to be fully infectious in permissive cells, and the modified E3 region or the inserted foreign gene was correctly expressed in the infected cells.

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.

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Liver toxicity and inflammation were assessed in C57BL/6, CBA, and BALB/c mice injected intravenously with a series of recombinant adenoviruses deleted simultaneously in E1/E3, in E1/E3/E2A, or in E1/E3/E4. All vectors were either devoid of transgenes or carried in E1 the human CFTR cDNA under the control of the CMV promoter. Injection of the E1/E3-deleted vector induced a significant liver dystrophy and inflammatory responses that were accompanied by an increased serum transaminase concentration. The vector toxicity remained elevated on additional deletion of the E2A gene and was further enhanced when hCFTR was expressed. In contrast, additional deletion of E4 led to a reduction in hepatotoxicity, suggesting an active role of E4 gene products in liver injury. However, deletion of E4 also led to a loss of transgene expression. To identify the individual E4 product(s) involved in liver toxicity and in the regulation of transgene expression, a series of isogenic E1/E3-deleted vectors, with or without the hCFTR transgene, and containing various combinations of functional E4 open reading frames (ORFs), were evaluated in vitro and in vivo. We demonstrate that liver injury was markedly reduced with vectors containing either ORF3 alone or ORF3,4 while vectors containing ORF4, ORF6,7 or ORF3,6,7 still displayed elevated hepatotoxicity and inflammatory responses. Moreover, transgene expression was restored when ORF3,4 or ORF3,6,7 was retained in the vector. These results highlight the importance of the E4 gene products in the design of improved in vivo gene transfer vectors.

Transgenic mice were generated in which 5 kb of the 5′ flanking promoter region of the human Factor IX (FIX) gene fused to various FIX constructs (gene, minigene and cDNA) were stably integrated in the germ line. Several transgenic mouse lines expressed high circulating levels of active and correctly processed recombinant human FIX. The presence of at least one FIX intron had a positive effect on the expression. The FIX transgenes were expressed in a tissue‐specific manner in the liver of transgenic mice. By crossing transgenic mice synthesizing FIX with others prone to develop hepatoma, progeny which co‐express the transgenes in hepatocytes were obtained. Hepatoma‐derived cell lines were shown to have a differentiated phenotype and secrete active human FIX for many generations.