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

Targeting the lung epithelium after intravenous delivery by directed evolution of underexplored sites on the AAV capsid


Advances in adeno-associated virus (AAV) engineering have provided exciting new tools for research and potential solutions for gene therapy. However, the lung has not received the same tailored engineering as other major targets of debilitating genetic disorders. To address this, here we engineered the surface-exposed residues AA452-458 of AAV9 capsid proteins at the three-fold axis of symmetry and employed a Cre-transgenic-based screening platform to identify AAV capsids targeted to the lung after intravenous delivery in mice. Using a custom image processing pipeline to quantify transgene expression across whole tissue images, we found that one engineered variant, AAV9.452sub.LUNG1, displays dramatically improved transgene expression in lung tissue after systemic delivery in mice. This improved transduction extends to alveolar epithelial type II cells, expanding the toolbox for gene therapy research for diseases specific to the lung.

Additional Information

© 2022 The Author(s). Under a Creative Commons license - Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). Received 22 February 2022, Accepted 15 July 2022, Available online 1 August 2022, Version of Record 9 August 2022. We thank the entire Gradinaru laboratory for helpful discussions. We thank Dr. Deverman for helpful discussions on CREATE design and implementation as described by Deverman and colleagues. We thank K. Johnston and P. Almhjell for assistance with development of the image processing pipeline and C. Oikonomou for assistance editing the manuscript. We are grateful to I. Antoshechkin and the Millar and Muriel Jacobs Genetics and Genomics Core at Caltech for assistance with next generation sequencing. We thank M. Smith for helpful discussions and assistance with the bioinformatics pipeline. This work was primarily supported by Defense Advanced Research Projects Agency grant W911NF-17-2-0036 to V.G. and by National Institutes of Health (NIH) to V.G.: NIH Pioneer DP1OD025535. D.G. was supported by the National Sciences and Engineering Research Council of Canada. N.G. was supported by the Cystic Fibrosis Foundation: CFF GOEDEN19F0. Author contributions. D.G. analyzed all data and prepared all figures with input from V.G., N.C.F., and N.G. designed and performed the variant selection experiments, D.G. characterized the variants and built the image processing pipeline. D.G. and V.G. wrote the manuscript with input from all authors. V.G. supervised all aspects of the work. Declaration of interests. The California Institute of Technology has filed and licensed patent applications for the work described in this manuscript, with N.C.F., N.G., and V.G. listed as inventors (US Patent application no. PCT/US21/46904). V.G. is a co-founder and board member and N.C.F. and N.G. are co-founders and officers of Capsida Biotherapeutics, a fully integrated AAV engineering and gene therapy company. The remaining authors declare no competing interests.

Attached Files

Published - 1-s2.0-S2329050122001024-main.pdf

Supplemental Material - 1-s2.0-S2329050122001024-mmc1.pdf


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August 22, 2023
December 22, 2023