Unlocking the secrets of gene therapy delivery: New insights into genome ejection from AAV vectors

Osaka, Japan - A research team at The University of Osaka has unveiled the molecular mechanism behind genome ejection from adeno-associated virus (AAV) vectors, a crucial delivery vehicle in gene therapy. The study reveals that the N-terminal region of the VP1 protein, a component of the AAV capsid, undergoes structural changes upon heating, facilitating the release of the therapeutic genetic material. This discovery offers new guidelines for vector design and stability assessment, promising more efficient and safer gene therapies.

AAV vectors are widely used in gene therapy to deliver therapeutic genes into target cells. However, the precise mechanism by which these vectors release their genetic cargo has remained elusive. The researchers investigated the relationship between structural changes in the viral protein VP1, crucial for endosomal escape, and genome release from adeno-associated virus (AAV) vectors. Using rAAV8, they prepared particles with varying VP1 content and VP3-only particles. By incrementally increasing temperature and employing techniques like mass photometry, nano-differential scanning fluorimetry, hydrogen/deuterium exchange mass spectrometry, and analytical ultracentrifugation, they observed capsid structural changes and genome ejection. A folded VP1 N-terminus hindered genome release, while unfolding facilitated it. Genome ejection occurred without capsid disintegration, resulting in three particle states: genome-containing, genome-tethered, and empty capsids.

This research provides a molecular-level understanding of how AAV vectors release their genomes, which is critical for designing more effective and safer gene therapies. The insights into VP1-mediated genome release will inform the development of improved vectors with enhanced delivery efficiency and reduced side effects. Senior author Susumu Uchiyama stresses the importance of these findings in overcoming current gene therapy challenges, "Gene therapy holds great promise as an innovative treatment for previously incurable diseases, but the production and quality control of vectors present significant challenges. Through meticulous analysis by our students and young faculty members, we have key indicators for stable AAV gene therapy vector production and storage, paving the way for delivering these innovative treatments to patients sooner."

###

The article, “Unfolding of viral protein 1 N-termini facilitates genome ejection from recombinant adeno-associated virus serotype 8,” was published in Molecular Therapy Methods & Clinical Development at DOI: https://doi.org/10.1016/j.omtm.2025.101480.

About The University of Osaka

The University of Osaka was founded in 1931 as one of the seven imperial universities of Japan and is now one of Japan's leading comprehensive universities with a broad disciplinary spectrum. This strength is coupled with a singular drive for innovation that extends throughout the scientific process, from fundamental research to the creation of applied technology with positive economic impacts. Its commitment to innovation has been recognized in Japan and around the world, being named Japan's most innovative university in 2015 (Reuters 2015 Top 100) and one of the most innovative institutions in the world in 2017 (Innovative Universities and the Nature Index Innovation 2017). Now, Osaka University is leveraging its role as a Designated National University Corporation selected by the Ministry of Education, Culture, Sports, Science and Technology to contribute to innovation for human welfare, sustainable development of society, and social transformation.

Website: https://resou.osaka-u.ac.jp/en