TY - JOUR
T1 - Comparative structural, biophysical, and receptor binding study of true type and wild type AAV2
AU - Bennett, Antonette
AU - Hull, Joshua
AU - Jolinon, Nelly
AU - Tordo, Julie
AU - Moss, Katie
AU - Binns, Enswert
AU - Mietzsch, Mario
AU - Hagemann, Cathleen
AU - Linden, R. Michael
AU - Serio, Andrea
AU - Chipman, Paul
AU - Sousa, Duncan
AU - Broecker, Felix
AU - Seeberger, Peter
AU - Henckaerts, Els
AU - McKenna, Robert
AU - Agbandje-McKenna, Mavis
N1 - Funding Information:
The TF20 cryo-electron microscope was provided by the UF College of Medicine (COM) and Division of Sponsored Programs (DSP). Data collection at Florida State University was made possible by NIH grants S10 OD018142-01 Purchase of a direct electron camera for the Titan-Krios at FSU (PI Taylor), S10 RR025080-01 Purchase of a FEI Titan Krios for 3-D EM (PI Taylor), and U24 GM116788 The Southeastern Consortium for Microscopy of MacroMolecular Machines (PI Taylor). The University of Florida COM and NIH GM082946 provided funds for the research efforts at the University of Florida. The work at KCL was sponsored by the King’s Commercialization Institute and a MRC grant (MR/S009302/1) awarded to EH.
Funding Information:
The TF20 cryo-electron microscope was provided by the UF College of Medicine (COM) and Division of Sponsored Programs (DSP). Data collection at Florida State University was made possible by NIH grants S10 OD018142-01 Purchase of a direct electron camera for the Titan-Krios at FSU (PI Taylor), S10 RR025080-01 Purchase of a FEI Titan Krios for 3-D EM (PI Taylor), and U24 GM116788 The Southeastern Consortium for Microscopy of MacroMolecular Machines (PI Taylor). The University of Florida COM and NIH GM082946 provided funds for the research efforts at the University of Florida. The work at KCL was sponsored by the King's Commercialization Institute and a MRC grant (MR/S009302/1) awarded to EH.
Publisher Copyright:
© 2021 Elsevier Inc.
PY - 2021/12
Y1 - 2021/12
N2 - 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.
AB - 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.
KW - AAV-TT
KW - Antibody binding
KW - Glycan array
KW - Receptor binding
KW - Stability
KW - cryo-EM
UR - http://www.scopus.com/inward/record.url?scp=85115798038&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85115798038&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2021.107795
DO - 10.1016/j.jsb.2021.107795
M3 - Article
C2 - 34509611
AN - SCOPUS:85115798038
SN - 1047-8477
VL - 213
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 4
M1 - 107795
ER -