Production of HF-GLAd with no RCA generation in the final product. (A) Production of HF-GLAd. GLAd genome (pGLAd_X) and helper plasmid (pAdBest_dITR) are co-transfected into a packaging cell line, HEK293T, in which the helper plasmid produces viral proteins for packaging of the GLAd genome. The helper plasmid cannot be amplified or packaged into the active viral particles, resulting in the production of HF-GLAd free of helper adenovirus. (B) No RCA generation during HF-GLAd production. Only one homologous recombination site is identified in the helper plasmid and E1-expressing packaging cells. Helper plasmid cannot acquire the E1 region from the genome of packaging cells via homologous recombination, which eliminates RCA generation in HF-GLAd production.