Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome.
Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome.
复制标题
使用细菌人工染色体中 ICP27 删除的超大 HSV-1 DNA 改进了 HSV 扩增子载体的辅助病毒包装系统。
DOI：
10.1006/mthe.2001.0294
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Chiocca,EA
中科院分区：
文献类型：
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作者：
Saeki,Y;Fraefel,C;Ichikawa,T;Breakefield,XO;Chiocca,EA
单纯疱疹病毒1型（HSV-1）扩增子是含有一个或多个转录单位和双作用HSV-1序列的原核质粒：DNA复制的病毒起源和病毒DNA切割/包装信号。在存在HSV-1“辅助”功能的情况下，扩增子被复制并包装成HSV-1病毒粒子。尽管最近包装方法有所改进，扩增子载体的库存仍然以10−4-10−6的频率被复制能力辅助病毒污染。为了克服这个问题，我们报告：(i)通过同源重组或位点特异性重组，可以在大肠杆菌中常规实现HSV-1基因组的遗传修饰；（ii）一种新的HSV-1细菌人工染色体（fHSVΔpacΔ27 0+），该染色体缺失了编码ICP27的必要基因，并增加了ICP0“填充”序列，使其长度增加到178 kb；支持共转染扩增子DNA的复制和包装，而不会产生复制能力强的辅助病毒（每108TU扩增子载体小于1个辅助病毒），并且（iii）所得扩增子库在浓缩后的滴度高达3-10 × 108TU/ml。从HSV-1扩增子载体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的安全性。
Herpes simplex virus type 1 (HSV-1) amplicons are prokaryotic plasmids containing one or more transcriptional units and twocis-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 inEscherichia coli, either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVΔpacΔ27 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 108TU amplicon vectors), and (iii) the resulting amplicon stocks have titers of up to 3–10 × 108TU/ml after concentration. Elimination of replication-competent helper virus from HSV-1 amplicon vector stocks further improves safety in gene transfer applications.