Helper virus-dependent production of GLAd and the generation of RCA in the final product. (A) Helper virus-dependent production of GLAd using the Cre/loxP system. Genomes of GLAd and helper adenovirus are co-transfected into packaging cells, Cre recombinase-expressing HEK293 cell line, in which both viruses are amplified and the helper adenovirus produces viral proteins. During this process, the ψ packaging signal flanked by loxP sites is excised by Cre recombinase, preventing the packaging of helper adenovirus genome, while the GLAd genome is packaged. The helper adenovirus contamination in the production of GLAd ranges from 0.01% to 10%. (B) The generation of RCA. Two homologous sites are identified in the genomes of E1-deleted helper adenoviruses and E1-expressing packaging cells. Homologous recombination can occur in these two homologous regions, resulting in the generation of RCA as the helper adenovirus acquires the E1 region from the packaging cells.
