A Novel Role for the Adenovirus L4 Region 22K and 33K Proteins in Adeno-Associated Virus Production

A pivotal article titled, “A novel role for the adenovirus L4 region 22K and 33K proteins in adeno-associated virus production,” was published by Forge experts in the peer-reviewed research journal Human Gene Therapy in January of 2024. The article was penned by team of scientists at Forge led by David Dismuke, Ph.D., Chief Technical Officer, Linas Padegimas, Ph.D., Molecular Development Senior Director, and Angela Adsero, Ph.D., Molecular Development Scientist II.

Key findings have the potential to advance the field of gene therapy. 

Specific adenoviral genes are required for AAV production. However, a novel, undiscovered gene in particular has been overlooked because of its shared DNA sequence with a gene regulatory region that determines when or how much protein is made from a gene. By using molecular techniques to decouple the dual-purpose nature of this sequence, the Forge scientific team demonstrated that the L4 region 22K protein is an additional requirement for AAV vector production that had previously gone unnoticed. The study also suggests that the L4 region 33K protein is important for increasing AAV production.

Read our full press release of the announcement.

An abstract follows, and you may access the full research article directly via Human Gene Therapy here.

Abstract
Despite decades of research in adeno-associated virus (AAV) and the role of adenovirus in production, the interplay of AAV and adenovirus is not fully understood. Specific regions of the adenoviral genome containing E1, E2a, E4 open reading frame (ORF), and VA RNA have been demonstrated as necessary for AAV production; however, incorporating these regions into either a producer cell line or subcloning into an Ad helper plasmid may lead to inclusion of neighboring adenoviral sequence or ORFs with unknown function. Because AAV is frequently used in gene therapies, removing excessive adenovirus sequences improves the Ad helper plasmid size and manufacturability, and may lead to safer vectors for patients. Furthermore, deepening our understanding of the helper virus genes required for recombinant AAV (rAAV) production has the potential to increase yields and manufacturability of rAAV for clinical and commercial applications. One region continuously included in various Ad helper plasmid iterations is the adenoviral E2a promoter region that appears to be necessary for E2a expression. Due to the compact nature of viral genomes, the E2a promoter region overlaps with the Hexon Assembly/100K protein and the L4 region. The L4 region, which contains the coding sequences for 22K and 33K proteins, had not been thought to be necessary for AAV production. Through molecular techniques, this study demonstrates that the adenoviral 22K protein is essential for rAAV production in HEK293 cells by triple transfection and that the 33K protein synergistically increases rAAV yield.