Saeki Y, Fraefel C, Ichikawa T, Breakefield X O, Chiocca E A
Molecular Neuro-Oncology Laboratories, Massachusetts General Hospital, Harvard Medical School, 149 13th Street, Massachusetts 02129, USA.
Mol Ther. 2001 Apr;3(4):591-601. doi: 10.1006/mthe.2001.0294.
Herpes simplex virus type 1 (HSV-1) amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis-acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal. In the presence of HSV-1 "helper" functions, amplicons are replicated and packaged into HSV-1 virions. Despite recent improvements in packaging methods, stocks of amplicon vectors are still contaminated with replication-competent helper virus at a frequency of 10(-4)-10(-6). To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli, either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVDeltapacDelta27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 "stuffer" sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected amplicon DNA without generating replication-competent helper virus (<1 helper virus per 10(8) TU amplicon vectors), and (iii) the resulting amplicon stocks have titers of up to 3-10 x 10(8) TU/ml after concentration. Elimination of replication-competent helper virus from HSV-1 amplicon vector stocks further improves safety in gene transfer applications.
1型单纯疱疹病毒（HSV-1）扩增子是原核质粒，包含一个或多个转录单元以及两个顺式作用的HSV-1序列：一个病毒DNA复制起点和一个病毒DNA切割/包装信号。在HSV-1“辅助”功能存在的情况下，扩增子被复制并包装到HSV-1病毒粒子中。尽管最近包装方法有所改进，但扩增子载体储备仍以10^(-4)-10^(-6)的频率被具有复制能力的辅助病毒污染。为克服这一问题，我们报道：（i）HSV-1基因组的基因修饰可通过同源重组或位点特异性重组在大肠杆菌中常规实现，（ii）一种新型的HSV-1细菌人工染色体（fHSVDeltapacDelta27 0+），其在编码ICP27的必需基因中有缺失，并添加了ICP0“填充”序列以使其大小增加到178 kb，可支持共转染的扩增子DNA的复制和包装，而不会产生具有复制能力的辅助病毒（每10^8个转导单位（TU）扩增子载体中<1个辅助病毒），并且（iii）浓缩后得到的扩增子储备滴度高达3-10×10^8 TU/ml。从HSV-1扩增子载体储备中消除具有复制能力的辅助病毒进一步提高了基因转移应用中的安全性。
