Expression of pIX gene induced by transgene promoter: Possible cause of host immune response in first-generation adenoviral vectors

First-generation (FG) adenoviral vectors (AdVs) have been widely used not only for gene therapy but also for basic studies. Because vectors of this type lack the E1A gene that is essential for the expression of other viral genes, their expression levels in target cells have been considered low. However, we found that the viral pIX gene, located immediately downstream of the inserted expression unit of the transgene, was significantly coexpressed with the transgene in cells infected with FG AdV. Whereas CAG and SR alpha promoters activated the pIX promoter considerably through their enhancer effects, the EF1 alpha promoter hardly did. Moreover, when the expression unit was inserted in the rightward orientation, not only the pIX protein but also a fusion protein consisting of the N-terminal part of transgene product and pIX were sometimes coexpressed with the transgene product through an aberrant splicing mechanism. In in vivo experiments, a LacZ-expressing AdV bearing the CAG promoter caused an elevation of alanine aminotransferase, but an AdV bearing the EF1 alpha promoter produced no detectable levels. Whereas the FG AdV expressing human growth hormone under the control of the CAG promoter maintained a high hormone level for less than 1 month, the FG AdV under the control of the EF1 alpha promoter maintained a high level for at least 6 months. These results suggest that pIX coexpression may be one of the main causes of AdV-induced immune responses, and that the EF1 alpha promoter is probably valuable for the long-term expression of FG AdV. Thus, the in vivo utility of FG AdV should be reevaluated.