E2A, VA RNA I, and L4-22k adenoviral helper genes are sufficient for AAV production in HEK293 cells.

The replication-defective adeno-associated virus (AAV) is extensively utilized as a research tool or vector for gene therapy. The production process of AAV remains intricate, expensive, and mechanistically underexplored. With the aim of enhancing AAV manufacturing efficiencies in mammalian cells, we revisited the questions and optimization surrounding the requirement of the various adenoviral helper genes in enabling AAV production. First, we refined the minimal set of adenoviral genes in HEK293 AAV production to , , and . These findings challenge the previously accepted necessity of adenoviral in AAV production. In addition, we identified genes as crucial helpers for AAV production. Next, a revised minimal adenoviral helper plasmid comprising , , and genes was designed and demonstrated to yield high titer and potent AAV preps in HEK293 transient transfection. Lastly, stable packaging cells harboring inducible and genes were shown to maintain AAV production yields comparable to transient transfection over a culture period of ∼10 weeks. Combined, these findings further our understanding of adenoviral helper function in mammalian AAV production and provide novel plasmid and cell-line reagents with an improved safety profile for potential broad applicability in the research and gene therapy community.