Structure of neurotropic adeno-associated virus AAVrh.8

Abstract

Adeno-associated virus rhesus isolate 8 (AAVrh.8) is a leading vector for the treatment of neurological diseases due to its efficient transduction of neuronal cells and reduced peripheral tissue tropism. Toward identification of the capsid determinants for these properties, the structure of AAVrh.8 was determined by X-ray crystallography to 3.5 Å resolution and compared to those of other AAV isolates. The capsid viral protein (VP) structure consists of an αA helix and an eight-stranded anti-parallel β-barrel core conserved in parvoviruses, and large insertion loop regions between the β-strands form the capsid surface topology. The AAVrh.8 capsid exhibits the surface topology conserved in all AAVs: depressions at the icosahedral twofold axis and surrounding the cylindrical channel at the fivefold axis, and three protrusions around the threefold axis. A structural comparison to serotypes AAV2, AAV8, and AAV9, to which AAVrh.8 shares ~84, ~91, and ~87% VP sequence identity, respectively, revealed differences in the surface loops known to affect receptor binding, transduction efficiency, and antigenicity. Consistent with this observation, biochemical assays showed that AAVrh.8 is unable to bind heparin and does not cross-react with conformational monoclonal antibodies directed against the other AAVs compared. This structure of AAVrh.8 thus identified capsid surface differences which can serve as template regions for rational design of vectors with enhanced transduction for specific tissues and escape pre-existing antibody recognition. These features are essential for the creation of an AAV vector toolkit that is amenable to personalized disease treatment.

Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging

“…6A). This additional density is proximal to but does not overlap with the position of the nucleotide density observed in the capsid interior of the majority of other wt-AAV serotype capsid structures determined to date (58). While a nucleotide model was not built into this 3-fold density, its absence in the AAV2-R432A is consistent with its DNA packaging defect.…”

Universal Method for the Purification of Recombinant AAV Vectors of Differing Serotypes

“…Notably, an AAV8-selective affinity resin was useful for the purification of a related serotype, AAVrh8R, while the performance of this serotype on an AAV9 selective resin was unsatisfactory. This result suggests that despite considerable sequence homology between AAV9 and AAVrh8R, 20 the epitope responsible for binding to the AAV9 resin is not shared; however, a common, as yet unidentified epitope facilitates binding of AAVrh8R to an AAV8-specific resin. These data are important, because they suggest that existing off-the-shelf serotype-specific affinity resins may be useful for the purification of disparate AAV capsids with conserved epitopes.…”

Adeno-Associated Virus (AAV) as a Vector for Gene Therapy

“…AAV has evolved to enter cells through initial interactions with carbohydrates present on the surface of target cells, typically sialic acid, galactose and heparin sulfate [ 29 , 30 ]. Subtle differences in sugar-binding preferences, encoded in capsid sequence differences, can influence cell-type transduction preferences of the various AAV variants [ 31 – 33 ]. For example, AAV9 has a preference for primary cell binding through galactose as a result of unique amino acid differences in its capsid sequence [ 34 ].…”