A DNA-based method to assay total and infectious particle contents and helper virus contamination in high-capacity adenoviral vector preparations.
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.
Center for Molecular Medicine (ZMMK), University of Cologne, 50931 Cologne, Germany.
Florian
F
Kreppel
Volker
V
Biermann
Stefan
S
Kochanek
Gudrun
G
Schiedner
D000256:Adenoviridae / Q000235:genetics* / Q000472:pathogenicity; D001681:Biological Assay / Q000379:methods*; D004279:DNA, Viral / Q000032:analysis; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000592:standards*; D006367:HeLa Cells; D006378:Helper Viruses / Q000302:isolation & purification*; D006801:Humans
D004279:DNA, Viral