Saeki Y, Ichikawa T, Saeki A, Chiocca E A, Tobler K, Ackermann M, Breakefield X O, Fraefel C
Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Charlestown 02129, USA.
Hum Gene Ther. 1998 Dec 10;9(18):2787-94. doi: 10.1089/hum.1998.9.18-2787.
Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only approximately 1% of the 152-kb HSV-1 genome, and consequently, replication and packaging into virions depends on helper functions. These helper functions have been provided conventionally by a helper virus, usually a replication-defective mutant of HSV-1, or more recently, by a set of five cosmids that overlap and represent the genome of HSV-1 deleted for DNA cleavage/packaging signals (pac). In the absence of pac signals, potential HSV-1 genomes that are reconstituted from the cosmids via homologous recombination are not packageable. The resulting amplicon stocks are, therefore, virtually free of contaminating helper virus. To simplify this packing system, the HSV-1 genome was cloned and maintained stably as a single-copy, F plasmid-based bacterial artificial chromosome in E. coli. Such a plasmid containing the HSV-1 genome deleted for the pac signals (fHSV delta pac) did not generate replication-competent progeny virus on transfection into mammalian cells, but rather, it was able to support the packaging of cotransfected amplicon DNA that contained a functional pac signal. The resulting amplicon vector stocks had titers of up to 10(7) transducing units per milliliter of culture medium and efficiently transduced neural cells in the rat brain, as well as hepatocytes in the rat. The capacity of generating infectious and replication-competent HSV-1 progeny following transfection into mammalian cells was restored after insertion of a pac signal into fHSV delta pac.
基于1型单纯疱疹病毒（HSV-1）的扩增子载体仅包含152kb HSV-1基因组的约1%，因此，其复制和包装成病毒粒子依赖于辅助功能。传统上，这些辅助功能由辅助病毒提供，通常是HSV-1的复制缺陷型突变体，或者最近由一组五个重叠的黏粒提供，这些黏粒代表缺失DNA切割/包装信号（pac）的HSV-1基因组。在没有pac信号的情况下，通过同源重组从黏粒中重建的潜在HSV-1基因组是不可包装的。因此，所得的扩增子储备实际上不含污染的辅助病毒。为了简化这种包装系统，HSV-1基因组被克隆并作为基于F质粒的单拷贝细菌人工染色体在大肠杆菌中稳定维持。这种含有缺失pac信号的HSV-1基因组的质粒（fHSV delta pac）在转染到哺乳动物细胞中时不会产生具有复制能力的子代病毒，相反，它能够支持共转染的含有功能性pac信号的扩增子DNA的包装。所得的扩增子载体储备在每毫升培养基中的滴度高达10^7转导单位，并能有效地转导大鼠脑中的神经细胞以及大鼠的肝细胞。在将pac信号插入fHSV delta pac后，转染到哺乳动物细胞中后产生有感染性和具有复制能力的HSV-1子代的能力得以恢复。
