An Improved Helper Plasmid Containing Deletions Within the E4 and E2a Genes Results in Increased Adeno-Associated...

The use of a helper plasmid to replace adenovirus infection for adeno-associated virus (AAV) manufacturing has been common practice for decades. Adenovirus E4, E2a, and VA RNA genes are sufficient to support efficient AAV replication. In an effort to ensure that all transfected DNA has a functional role in AAV production, deletions were introduced to the E4 and E2a genes to determine if any portions were dispensable. Although a 900 bp deletion in the E2a intron did not have an impact, the removal of open reading frames (orf) 1-4 from the E4 gene resulted in a doubling of AAV productivity. The E4Δorf1-4 deletion was associated with a reduction in E4orf6 transcripts, along with an increase in Rep and Cap transcripts and protein levels, which corresponded to increased AAV productivity in crude lysate. The final product of these studies was a helper plasmid, termed OXB-Helper_3, that is >3.4 kb smaller than the original control plasmid and resulted in ∼2× improvement in vector genome productivity across multiple capsid serotypes, genome designs, and transfection platforms.