Engineered Sleeping Beauty Transposon as Efficient System to Optimize Chimp Adenoviral Production

: Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors.