Modulation of the inflammatory properties and hepatotoxicity of recombinant adenovirus vectors by the viral E4 gene products

Liver toxicity and inflammation were assessed in C57BL/6, CBA, and BALB/c m ice injected intravenously with a series of recombinant adenoviruses delete d simultaneously in E1/E3, in E1/E3/E2A, or in E1/E3/E4. All vectors were e ither devoid of transgenes or carried in El the human CFTR cDNA under the c ontrol of the CMV promoter. Injection of the E1/E3-deleted vector induced a significant liver dystrophy and inflammatory responses that were accompani ed by an increased serum transaminase concentration. The vector toxicity re mained elevated on additional deletion of the E2A gene and was further enha nced when hCFTR was expressed, In contrast, additional deletion of E4 led t o a reduction in hepatotoxicity, suggesting an active role of E4 gene produ cts in liver injury, However, deletion of E4 also led to a loss of transgen e expression. To identify the individual E4 product(s) involved in liver to xicity 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 eval uated in vitro and in vivo, We demonstrate that liver injury was markedly r educed with vectors Containing either ORF3 alone or ORF3,4 while vectors co ntaining ORF4, ORF6,7 or ORF3,6,7 still displayed elevated hepatotoxicity a nd 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.