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Human Gene Therapy
A DNA-Based Method to Assay Total and Infectious Particle Contents and Helper Virus Contamination in High-Capacity Adenoviral Vector Preparations
To cite this article:
Florian Kreppel, Volker Biermann, Stefan Kochanek, Gudrun Schiedner.
Human Gene Therapy.
July 2002,
13(10): 1151-1156.
doi:10.1089/104303402320138934.
Florian Kreppel Center for Molecular Medicine (ZMMK), University of Cologne, 50931 Cologne, Germany. Volker Biermann Center for Molecular Medicine (ZMMK), University of Cologne, 50931 Cologne, Germany. Stefan Kochanek Center for Molecular Medicine (ZMMK), University of Cologne, 50931 Cologne, Germany. Gudrun Schiedner Center for Molecular Medicine (ZMMK), University of Cologne, 50931 Cologne, Germany. 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.  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