Ruben Hernandez‐Alcoceba

Background and Aims High‐capacity adenoviral vectors (HC‐AdV) show extended DNA payload and stability of gene expression in vivo due to the absence of viral coding sequences. However, production requires methods to trans‐complement viral proteins, usually through Helper Viruses (HV). The Cre/loxP system is frequently employed to remove the packaging signal in HV genomes, in order to avoid their encapsidation. However, chronic exposure to the Cre recombinase in packaging cells is detrimental. We have applied the dimerizable Cre system to overcome this limitation. Methods and Results Cre was split in two fragments devoid of recombinase function (N‐terminal 244 and C‐terminal 99 amino‐acids). In one version of the system, interaction with both moieties was favored by rapamycin‐dependent heterodimerization domains (DiCre). Other version contained only Cre sequences (oCre). We generated packaging cells and HVs expressing the complementary fragments and studied their performance for HC‐AdV production. We found that both conformations avoided interference with the growth of packaging cells, and the oCre system was particularly suitable for HC‐AdV amplification. Conclusions The split‐Cre system improves the performance of packaging cells and can reduce the time and cost of HC‐AdV amplification up to 30% and 15%, respectively. This may contribute to the standardization of HC‐AdV production.