A DNA-based method to assay total and infectious particle contents and helper virus contamination in high-capacity adenoviral vector preparations

被引:68
作者
Kreppel, F [1 ]
Biermann, V [1 ]
Kochanek, S [1 ]
Schiedner, G [1 ]
机构
[1] Univ Cologne, Ctr Mol Med ZMMK, D-50931 Cologne, Germany
关键词
D O I
10.1089/104303402320138934
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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.
引用
收藏
页码:1151 / 1156
页数:6
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