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Published September 8, 2022 | Supplemental Material + Published
Journal Article Open

Structural basis of receptor usage by the engineered capsid AAV-PHP.eB

Abstract

Adeno-associated virus serotype 9 (AAV9) is a promising gene therapy vector for treating neurodegenerative diseases due to its ability to penetrate the blood-brain barrier. PHP.eB was engineered from AAV9 by insertion of a 7-amino acid peptide and point mutation of neighboring residues, thereby enhancing potency in the central nervous system. Here, we report a 2.24-Å resolution cryo-electron microscopy structure of PHP.eB, revealing conformational differences from other 7-mer insertion capsid variants. In PHP.eB, the 7-mer loop adopts a bent conformation, mediated by an interaction between engineered lysine and aspartate residues. Further, we identify PKD2 as the main AAV receptor (AAVR) domain recognizing both AAV9 and PHP.eB and find that the PHP.eB 7-mer partially destabilizes this interaction. Analysis of previously reported AAV structures together with our pull-down data demonstrate that the 7-mer topology determined by the lysine-aspartate interaction dictates AAVR binding strength. Our results suggest that PHP.eB's altered tropism may arise from both an additional interaction with LY6A and weakening of its AAVR interaction. Changing the insertion length, but not sequence, modifies PKD2 binding affinity, suggesting that a steric clash impedes AAVR binding. This research suggests improved library designs for future AAV selections to identify non-LY6A-dependent vectors and modulate AAVR interaction strength.

Additional Information

© 2022 The Author(s). Published by Elsevier. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Received 29 April 2022, Accepted 15 July 2022, Available online 31 July 2022. We sincerely thank the staff of the Caltech CLARITY, Optogenetics, & Vector Engineering Center (CLOVER), and members of the Gradinaru lab, especially Josette Medicielo and Zhe Qu, for their help with virus purification, and Catherine Oikonomou for help editing the manuscript. Cryo-EM was done in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. We especially thank Songye Chen for assisting with cryo-EM data collection. We are grateful to all members of phylogeny.fr and particularly Sebastien Santini (CNRS/AMU IGS UMR7256) and the PACA BioInfo platform (supported by IBiSA) for the availability and management of phylogeny.fr. This work is supported by a grant from the National Institutes of Health (NIH) to V.G. (NIH Pioneer DP1OD025535). Figure 3A was created with BioRender.com. Author contributions: S.J., T.F.M., and V.G. conceived the idea, and V.G. supervised this project. S.J. determined the cryo-EM structures. S.J. and H.K.S. purified the capsids and proteins. S.J. and H.K.S. conducted the pull-down assays. X.D. conducted the RosettaRemodel prediction. S.J., T.F.M., and V.G. wrote the manuscript, and all authors reviewed the data and manuscript. The authors declare no competing interests. Data availability statement: Cryo-EM map and 3D coordinates are available in wwPDB and EMDB with the following accession codes: I1 symmetry applied AAV-PHP.eB, PDB: 7UD4 and EMD: 26453 and symmetry-free AAV-PHP.eB, EMD: 26417.

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Published - 1-s2.0-S2329050122001036-main.pdf

Supplemental Material - 1-s2.0-S2329050122001036-mmc1.pdf

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Additional details

Created:
August 22, 2023
Modified:
December 22, 2023