Abstract
High-capacity adenovirus (HC-Ad) vectors have been developed to address capacity, toxicity, and immunogenicity problems of first- and second-generation adenovirus vectors. HC-Ad vectors cannot be produced similar to helper-independent vectors, in which most viral functions are provided from the vector. Since adenovirus is a relatively large DNA virus that expresses many different protein and RNA functions, it is unlikely that complementing cell lines can be generated to provide appropriate levels of all viral functions in “trans.” The DNA-based method described here allows for fast and reliable determination of all three parameters with standard laboratory equipment independent of viral or reporter gene expression. The production process of HC-Ad vectors builds on two viral components; vector and El-deleted helper virus with packaging signal flanked by loxP sites and serial amplifications in producer cells expressing Crerecombinase. Excision of the packaging signal from the helper virus in the producer cells results in preferential packaging of vector genomes and a reduction of helper virus contamination.
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