Adeno-associated virus capsid assembly is divergent and stochastic

• Published in: Nature communications 2021-03, Vol.12 (1), p.1642-1642, Article 1642
• Main Authors: Wörner, Tobias P., Bennett, Antonette, Habka, Sana, Snijder, Joost, Friese, Olga, Powers, Thomas, Agbandje-McKenna, Mavis, Heck, Albert J. R.
• Format: Article
• Language: English
• Subjects: 631/1647/296; 631/57/2272/2276; 82/58; Animals; Capsid - chemistry; Capsid - metabolism; Capsid Proteins - chemistry; Capsid Proteins - metabolism; Capsids; Composition; Dependovirus - genetics; Dependovirus - metabolism; Expression vectors; Gene therapy; Genomes; HEK293 Cells; High resolution; Humanities and Social Sciences; Humans; Icosahedral phase; Mass spectra; Mass spectrometry; Mass spectroscopy; multidisciplinary; N-Terminus; Proteins; Science; Science (multidisciplinary); Serogroup; Serotypes; Sf9 Cells; Stochasticity; Stoichiometry; Vectors (Biology); Viral Proteins - metabolism; Virus Assembly; Viruses
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-21935-5

Adeno-associated viruses (AAVs) are increasingly used as gene therapy vectors. AAVs package their genome in a non-enveloped T  = 1 icosahedral capsid of ~3.8 megaDalton, consisting of 60 subunits of 3 distinct viral proteins (VPs), which vary only in their N-terminus. While all three VPs play a role in cell-entry and transduction, their precise stoichiometry and structural organization in the capsid has remained elusive. Here we investigate the composition of several AAV serotypes by high-resolution native mass spectrometry. Our data reveal that the capsids assemble stochastically, leading to a highly heterogeneous population of capsids of variable composition, whereby even the single-most abundant VP stoichiometry represents only a small percentage of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition. The systematic scoring of the simulations against experimental native MS data offers a sensitive new method to characterize these therapeutically important heterogeneous capsids. Adeno-associated viruses (AAVs) have emerged as promising gene therapy vectors.The AAV capsid consists of 60 subunits made up from three distinct viral proteins (VPs). Here authors record high-resolution native mass spectra of intact AAV capsids to assess the VP stoichiometries in a panel of serotypes and reveals an extremely heterogeneous population of capsids of variable composition.