An engineered helper plasmid generates differential E4orf6 and L4-22/33K gene expression increasing AAV vector...

Helper plasmids that depend on native adenovirus gene expression have long been the standard for transient adeno-associated virus (AAV) production. Here, we demonstrate that engineering the required helper gene expression can greatly increase AAV production relative to the use of native adenovirus gene regulation. Two different engineered helper plasmid designs improved AAV vector genome (VG) titers up to 5-fold compared to a standard helper plasmid. A substantial decrease in adenovirus E4orf6 and an increase in L4 22K and 33K gene expression were associated with the improved engineered helper plasmids. VG titer improvement across capsid serotypes, plasmid transfection platforms and genome designs suggest that engineering helper gene expression is widely suited to improving AAV manufacturing yields while also maintaining consistent vector quality attributes.