Comparative Analysis of the Capsid Structures of AAVrh.10, AAVrh.39, and AAV8

Abstract

Adeno-associated viruses (AAVs) from clade E are often used as vectors in gene delivery applications. This clade includes rhesus isolate 10 (AAVrh.10) and 39 (AAVrh.39) which, unlike representative AAV8, are capable of crossing the blood-brain barrier (BBB), thereby enabling the delivery of therapeutic genes to the central nervous system. Here, the capsid structures of AAV8, AAVrh.10 and AAVrh.39 have been determined by cryo-electron microscopy and three-dimensional image reconstruction to 3.08-, 2.75-, and 3.39-Å resolution, respectively, to enable a direct structural comparison. AAVrh.10 and AAVrh.39 are 98% identical in amino acid sequence but only ∼93.5% identical to AAV8. However, the capsid structures of all three viruses are similar, with only minor differences observed in the previously described surface variable regions, suggesting that specific residues S269 and N472, absent in AAV8, may confer the ability to cross the BBB in AAVrh.10 and AAVrh.39. Head-to-head comparison of empty and genome-containing particles showed DNA ordered in the previously described nucleotide-binding pocket, supporting the suggested role of this pocket in DNA packaging for the Dependoparvovirus. The structural characterization of these viruses provides a platform for future vector engineering efforts toward improved gene delivery success with respect to specific tissue targeting, transduction efficiency, antigenicity, or receptor retargeting. IMPORTANCE Recombinant adeno-associated virus vectors (rAAVs), based on AAV8 and AAVrh.10, have been utilized in multiple clinical trials to treat different monogenetic diseases. The closely related AAVrh.39 has also shown promise in vivo. As recently attained for other AAV biologics, e.g., Luxturna and Zolgensma, based on AAV2 and AAV9, respectively, the vectors in this study will likely gain U.S. Food and Drug Administration approval for commercialization in the near future. This study characterized the capsid structures of these clinical vectors at atomic resolution using cryo-electron microscopy and image reconstruction for comparative analysis. The analysis suggested two key residues, S269 and N472, as determinants of BBB crossing for AAVrh.10 and AAVrh.39, a feature utilized for central nervous system delivery of therapeutic genes. The structure information thus provides a platform for engineering to improve receptor retargeting or tissue specificity. These are important challenges in the field that need attention. Capsid structure information also provides knowledge potentially applicable for regulatory product approval.

Characterization of AAV-Specific Affinity Ligands: Consequences for Vector Purification and Development Strategies

“…However, structural comparison of empty and full AAV8 capsids complexed with AVB or CSAL8 reconstructed to 2.7–2.8 Å resolution did not show major differences on the capsid surfaces explaining the observed binding difference (data not shown). Similar to the recent study on the structures of genome-containing and empty AAV8 capsids, 21 there were differences on the inside of the capsid. Thus, a different factor, possibly the isoelectric point (pI), could serve as the determinant of this differential recognition, a concept discussed previously for separation of empty and genome-containing capsids.…”

“…The capsid structures of numerous wild-type AAVs and capsid variants are available. 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 The T = 1 icosahedral capsids are composed of 60 viral proteins (VPs) assembled from VP1 (~80 kDa), VP2 (~65 kDa), and VP3 (~60 kDa), in an approximate ratio of 1:1:10, respectively. 29 The VPs consist of a conserved α helix (αA), a βA strand, and an eight-stranded antiparallel β barrel (βB-βI) core motif connected by large loops, named after the β strands that they connect.…”

Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking

“…This reversibility indicates that the DNA density was either compressed or became disordered due to lack of VP interactions at low pH and left a gap between the capsid interior and packaged genome. Furthermore, in DNA-filled AAV capsids, a space exists under the icosahedral 5-fold axes [ 48 ]. Evidence suggests that the viral genome is ejected from intact parvovirus capsids in a 3′-5′ direction via the 5-fold channel in parvovirus MVM and the AAVs [ 49 , 50 ].…”

Structural Characterization of Cuta- and Tusavirus: Insight into Protoparvoviruses Capsid Morphology

“…The less ordered state in this region of the TuV map could be the result of the presence of multiple smaller amino acids 531-GAAV-534 conferring flexibility to this loop. The final VP models, refined in the context of their respective 60mers relative to the cryo-reconstructed maps, had high CC and good geometry values comparable to other virus structures determined to similar resolution ( Table 1 ) [ 9 , 33 , 35 , 36 , 37 ].…”