Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Microbiol Immunol. 2012 Jul;56(7):447-55. doi: 10.1111/j.1348-0421.2012.00454.x.
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.
第一代腺病毒（AdV）能够实现高效的基因转导，但它的免疫原性是体内的一个重要问题。辅助依赖性 AdV（HD-AdV）是解决这个问题的一种可能方法。HD-AdV 的构建需要一种辅助病毒，在这种病毒中，病毒包装结构域的两侧插入了两个loxP，以阻止其在 Cre 表达的 293 细胞中包装。在这里，我们构建了含有基因组包装结构域上游 191nt 处loxP 的 19L 病毒、含有 143nt 处loxP 的 15L 病毒和一个没有这些位置loxP 的对照ΔL 病毒。19L 位置在全球范围内使用，而 15L 位置已被报道会导致滴度低于 19L。当比较六对 19L 和 15L AdV 的滴度时，与之前的报道结果不同，19L AdV 的滴度与 15L 和ΔL AdV 相似，有时甚至低于 15L 和ΔL AdV。接下来，我们选择了一对滴度与ΔL 相似的 15L 和 19L AdV，以及一个缺乏loxP 的竞争 AdV，用于竞争实验。当少量竞争 AdV 共同感染时，只有 15L 和 19L AdV，但不是ΔL，在随后的病毒传代中逐渐成为少数成分。因此，143nt 和 191nt 处的loxP 插入降低了病毒的包装效率。
