A DNA-based method to assay total and infectious particle contents and helper virus contamination in high-capacity adenoviral vector preparations
A DNA-based method to assay total and infectious particle contents and helper virus contamination in high-capacity adenoviral vector preparations
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DOI：
10.1089/104303402320138934
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发表时间：
2002-07-01
影响因子：
4.2
通讯作者：
Schiedner, G
中科院分区：
文献类型：
--
作者：
Kreppel, F;Biermann, V;Schiedner, G
高容量腺病毒（HC-Ad）载体缺乏所有病毒基因。因此，这些载体具有降低毒性、免疫原性和增加外源DNA容量的特点。HC-Ad载体是在e1转化细胞系中产生的，在e1缺失的辅助病毒的存在下产生的，该辅助病毒在反式中提供了产生载体所需的所有病毒功能。通过cre/loxP-或FLPe/ frt介导的重组，在载体生产过程中切除辅助病毒的包装信号，产生不可包装的辅助病毒基因组。虽然重组酶介导的包装信号从辅助病毒基因组中切除是非常有效的，但仍有少数保留包装信号的辅助病毒基因组被包装到衣壳中。在临床试验中，HC-Ad载体制剂必须根据(1)总HC-Ad载体颗粒的数量，(2)感染性HC-Ad载体颗粒的数量，以及(3)污染辅助病毒颗粒的数量来准确表征。我们描述了一个快速和通用的dna为基础的生物测定这三个参数的标准实验室方法。该试验是确定腺病毒载体制剂生物活性数据的有用工具，重要的是，允许它们在不同研究之间进行比较。
High-capacity adenoviral (HC-Ad) vectors are devoid of all viral genes. Therefore, these vectors feature reduced toxicity, immunogenicity, and increased capacity for foreign DNA. HC-Ad vectors are produced in E1-transformed cell lines in the presence of an E1-deleted helper virus that provides in trans all viral functions necessary for vector production. By cre/loxP- or FLPe/Frt-mediated recombination the packaging signal of the helper virus is excised during vector production resulting in nonpackagable helper virus genomes. Although recombinase-mediated excision of the packaging signal from the helper virus genome is highly efficient, a small number of helper virus genomes with retained packaging signals are still packaged into capsids. For clinical trials, HC-Ad vector preparations have to be characterized accurately with respect to the number of (1) total HC-Ad vector particles, (2) infectious HC-Ad vector particles, and (3) the number of contaminating helper virus particles. We describe a fast and versatile DNA-based biologic assay for determination of these three parameters by standard laboratory methods. This assay is a useful tool for determining bioactivity data of adenoviral vector preparations and, importantly, allows their comparison among different studies.