Influence of loxP insertion upstream of the cis-acting packaging domain on adenovirus packaging efficiency

First-generation AdV enables efficient gene transduction, although its immunogenicity is an important problem in vivo. Helper-dependent AdV (HD-AdV) is one possible solution to this problem. The construction of HD-AdV requires a helper virus, in which the viral packaging domain is flanked by two inserted loxP to hamper its packaging in Cre-expressing 293 cells. Here, we constructed 19L viruses containing loxP at 191 nt from the left end of the genome upstream of the packaging domain, 15L viruses bearing loxP at 143 nt, and a control ΔL virus lacking loxP at these positions. The 19L position is used worldwide, and the 15L position has been reported to result in a lower titer than that of 19L. When the titers were compared for six pairs of 19L and 15L AdV, the 19L AdV produced titers similar to, or sometimes lower than, the 15L and ΔL AdV, unlike the results of previous reports. We next chose one pair of 15L and 19L AdV that produced titers similar to that of ΔL and a competitor AdV lacking loxP for use in a competition assay. When a small amount of the competitor AdV was co-infected, both the 15L and the 19L AdV, but not ΔL, gradually became minority components during subsequent viral passages. Therefore, the loxP insertions at 143 nt and 191 nt decreased the viral packaging efficiency.