Gonçalves Manuel A F V, van der Velde Ietje, Knaän-Shanzer Shoshan, Valerio Dinko, de Vries Antoine A F
Gene Therapy Section, Department of Molecular Cell Biology, Leiden University Medical Center, 2333 AL Leiden, The Netherlands.
Virology. 2004 Apr 10;321(2):287-96. doi: 10.1016/j.virol.2004.01.007.
Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors.
为了使用大DNA分子对靶细胞进行高效且永久的基因改造，需要具有高克隆能力和宿主染色体整合能力的病毒载体。我们构建了一种杂交基因传递载体，它由包装在腺病毒（Ad）衣壳中的重组腺相关病毒（AAV）复制中间体组成。这种组合使得重组AAV基因组能够在不依赖细胞周期的情况下进入细胞核，其长度大大超过了AAV衣壳所能容纳的最大尺寸（即4.7 kb）。在这里，我们表明高容量AAV/Ad杂交载体基因传递介导了大片段外源DNA的细胞基因组整合，并在快速增殖的细胞中实现了稳定的长期转基因表达。对从稳定转导细胞克隆中提取的染色体DNA进行Southern印迹和聚合酶链反应分析表明，大多数克隆都包含一个全长杂交载体基因组的单拷贝，两端带有AAV反向末端重复（ITR）序列。因此，高容量AAV/Ad杂交载体系统可用于转移和表达传统整合病毒载体无法传递的转基因。
