Comparative structural, biophysical, and receptor binding study of true type and wild type AAV2

Antonette Bennett, Joshua Hull, Nelly Jolinon, Julie Tordo, Katie Moss, Enswert Binns, Mario Mietzsch, Cathleen Hagemann, R. Michael Linden, Andrea Serio, Paul Chipman, Duncan Sousa, Felix Broecker, Peter Seeberger, Els Henckaerts*, Robert McKenna, Mavis Agbandje-McKenna

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

Abstract

Adeno-associated viruses (AAV) are utilized as gene transfer vectors in the treatment of monogenic disorders. A variant, rationally engineered based on natural AAV2 isolates, designated AAV-True Type (AAV-TT), is highly neurotropic compared to wild type AAV2 in vivo, and vectors based on it, are currently being evaluated for central nervous system applications. AAV-TT differs from AAV2 by 14 amino acids, including R585S and R588T, two residues previously shown to be essential for heparan sulfate binding of AAV2. The capsid structures of AAV-TT and AAV2 visualized by cryo-electron microscopy at 3.4 and 3.0 Å resolution, respectively, highlighted structural perturbations at specific amino acid differences. Differential scanning fluorimetry (DSF) performed at different pH conditions demonstrated that the melting temperature (Tm) of AAV2 was consistently ∼5 °C lower than AAV-TT, but both showed maximal stability at pH 5.5, corresponding to the pH in the late endosome, proposed as required for VP1u externalization to facilitate endosomal escape. Reintroduction of arginines at positions 585 and 588 in AAV-TT caused a reduction in Tm, demonstrating that the lack of basic amino acids at these positions are associated with capsid stability. These results provide structural and thermal annotation of AAV2/AAV-TT residue differences, that account for divergent cell binding, transduction, antigenic reactivity, and transduction of permissive tissues between the two viruses. Specifically, these data indicate that AAV-TT may not utilize a glycan receptor mediated pathway to enter cells and may have lower antigenic properties as compared to AAV2.

Original languageEnglish
Article number107795
JournalJournal of Structural Biology
Volume213
Issue number4
DOIs
Publication statusPublished - Dec 2021

Keywords

  • AAV-TT
  • Antibody binding
  • cryo-EM
  • Glycan array
  • Receptor binding
  • Stability

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