Structure comparison of the chimeric AAV2.7m8 vector with parental AAV2

Abstract

The AAV2.7m8 vector is an engineered capsid with a 10-amino acid insertion in Adeno-associated virus (AAV) surface variable region VIII (VR-VIII) resulting in the alteration of an antigenic region of AAV2 and the ability to efficiently transduce retina cells following intravitreal administration. Directed evolution and in vivo screening in the mouse retina isolated this vector. In the present study, we sought to identify the structural differences between a recombinant AAV2.7m8 (rAAV2.7m8) vector packaging a GFP genome and its parental serotype, AAV2, by cryo-electron microscopy (cryo-EM) and image reconstruction. The structures of rAAV2.7m8 and AAV2 were determined to 2.91 and 3.02 Å resolution, respectively. The rAAV2.7m8 amino acid side-chains for residues 219-745 (the last C-terminal residue) were interpretable in the density map with the exception of the 10 inserted amino acids. While observable in a low sigma threshold density, side-chains were only resolved at the base of the insertion, likely due to flexibility at the top of the loop. A comparison to parental AAV2 (ordered from residues 217-735) showed the structures to be similar, except at some side-chains that had different orientations and, in VR-VIII containing the 10 amino acid insertion. VR-VIII is part of an AAV2 antigenic epitope, and the *

Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders

“…This loop forms the second highest peak at the threefold symmetry axis of the AAV2 capsid and is part of the HSPG‐binding motif as well as a target of neutralising antibodies. Similar to our novel capsids, the insertion of LALGETTRP peptide in AAV2.7m8 extended this loop and changed its structure (Bennett et al , 2020). Based on our in silico modelling, the loop extensions of both novel variants appear to be oriented in the opposite direction compared with AAV2.7m8.…”

“…Key residues N587 and R588 shown in green. E, FFocused view on the variable loops 3 and 4 of the comparative overlaps in (C) and (D), respectively. The colour scheme is consistent as before. G, HFocused view on the variable loops 3 and 4 of the comparative overlap of structural monomers AAV2.GL and AAV2.NN, respectively, with AAV2.7m8 (PDB 6U0R) (Bennett et al , 2020). The colour scheme for AAV2.GL (G) and AAV2.NN (H) is as before.…”

“…Based on our in silico modelling, the loop extensions of both novel variants appear to be oriented in the opposite direction compared with AAV2.7m8. This however is subject to confirmation via structural analysis, as recent work indicated that such peptide‐extended loops can be flexible in 3D space (Bennett et al , 2020).…”

Completion of the AAV Structural Atlas: Serotype Capsid Structures Reveals Clade-Specific Features

“…The AAV7, AAV11–13 VP topologies conserve the core eight-stranded anti-parallel β-barrel (βB-βI), with the additional β-strand A and α-helix A ( Figure 4 A), as described previously for all other AAV structures [ 9 , 11 , 12 , 13 , 14 , 15 , 17 , 18 , 19 , 46 , 55 , 56 , 57 , 58 , 59 ]. When superposed, these core regions are homologous for the AAV serotypes ( Figure 4 A).…”

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

“…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.…”