Adeno-associated virus capsid assembly is divergent and stochastic

Abstract

Adeno-associated viruses (AAVs) are small, non-enveloped, and have a T=1 icosahedral capsid. They belong to the Parvoviridae, genus Dependoparvovirus. Interest in AAVs has grown over recent years as they have emerged as promising gene therapy vectors. The AAV capsid, encapsulating the transgene, consists, in total, of 60 subunits made up from three distinct viral proteins (VPs) originating from the same cap gene (VP1, VP2, and VP3), which vary only in their N-terminus. While all three VPs play a crucial and specific role in cell-entry and transduction, their exact stoichiometry and organization in AAV capsids has, despite the availability of several high-resolution structures remained elusive. Here we obtained a set of native mass spectra of intact AAV capsids (Mw ≈ 3.8 MDa) that display both highly resolved regions and regions wherein interferences occur. Through spectrum simulation we resolved and elucidated this spectral complexity, allowing us to directly assess the VP stoichiometries in a panel of serotypes from different production platforms. The data reveals an extremely heterogeneous population of capsids of variable composition. The relative abundance for each of the hundreds of co-occurring capsid compositions is accurately described by a model based upon stochastic assembly from a mixed pool of expressed VP1, VP2, and VP3. We show that even the single-most abundant VP stoichiometry represents only a few percent of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition and arrangement, i.e. no particle is identical. The systematic scoring of the stochastic assembly model against experimental high-resolution native MS data offers a sensitive and accurate new method to characterize these exceptionally heterogeneous gene-delivery vectors.

Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification

“…Development of HPLC assays for adeno-associated virus (AAV) must accommodate a more challenging reality. Individual AAV capsids comprise 60 capsid proteins of 3 types, ideally but often not in a ratio of 1:1:10; which may lack encapsidated DNA or contain DNA of varying lengths, with varying proportions of single-stranded and double-stranded sequences [ 1 , 2 , 3 ]. Post-translational modifications impose additional layers of heterogeneity [ 3 , 4 , 5 ].…”

“…Individual AAV capsids comprise 60 capsid proteins of 3 types, ideally but often not in a ratio of 1:1:10; which may lack encapsidated DNA or contain DNA of varying lengths, with varying proportions of single-stranded and double-stranded sequences [ 1 , 2 , 3 ]. Post-translational modifications impose additional layers of heterogeneity [ 3 , 4 , 5 ]. AAV cell harvests and lysates bear contaminant species and levels similar to those encountered with recombinant protein cell culture harvests but AAV levels are orders of magnitude lower, leaving AAV virtually a trace component in an overwhelming excess of contaminants [ 6 ].…”

Multiple-parameter profiling of density gradient ultracentrifugation for characterization of empty and full capsid distribution in AAV preparations

“…Recent studies have reported that capsid f FIGURE 5 Centrifugram of the empty capsid peak from AEX after AEX of CEX-purified AAV. composition frequently varies from the ideal virus protein ratios of 1 VP1 to 1 VP2 to 10 VP3 [24], and that post-translational deamidation can substantially alter capsid surface charge [25]. The ramp region might contain such variants.…”

Anion-exchange HPLC assay for separation and quantification of empty and full capsids in multiple adeno-associated virus serotypes

“…To date, a variety of methods have been used to quantify empty and full AAV capsids, which include UV/Vis spectroscopy and capsid (cp)-to-vector genome (vg) titer ratio, 8 transmission electron microscopy (TEM), 9 analytical ultracentrifugation (AUC), 10 size exclusion chromatography coupled to multiangle light scattering (SEC-MALS), 11 capillary electrophoresis, 12 mass photometry, 13 and mass spectrometry (MS). [14][15][16] Each method utilizes a unique approach toward addressing the quantification of the empty-to-full ratio of an AAV capsid. Each method, however, also has shortcomings.…”