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Published June 8, 2023 | Published + Supplemental Material
Journal Article Open

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates


Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds, and in rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and in ex vivo human brain slices, although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial-specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. We apply this approach to Hevin knockout mice, where AAV-X1-mediated ectopic expression of the synaptogenic protein Sparcl1/Hevin in brain endothelial cells rescued synaptic deficits.

Additional Information

© The Author(s) 2023. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. We thank members of the Gradinaru group for their assistance in this study: Yaping Lei for help with virus production, Miguel Chuapoco for discussion on the macaque experiment, Elisha Mackey for mouse colony management, Zhe Qu for lab management, Patricia Anguiano for administrative assistance, and the entire Gradinaru group for discussions. We thank I. Antoshechkin and the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech for providing sequencing services. We thank the Beckman Institute Single-Cell Profiling and Engineering Center (SPEC) at Caltech and Sisi Chen for providing sequencing machines. We thank Annie W. Lam and Jost Vielmetter of the Beckman Institute Protein Expression Center at Caltech for Ly6a protein production and for providing surface plasmon resonance machines. We thank Sian Murphy at SWC's Neurobiological Research Facility (NRF) for the injection of the rats. We thank Cassandra Tang-Wing and Chris Chamberlain at UCSD for the injection of the marmosets. We thank Michael Metke and Vikram Pal Singh at UCSD for the tissue collection from marmosets. We thank the staff at the California National Primate Research Center for the experiment in rhesus macaque. We thank Catherine Oikonomou for help with manuscript editing. This work was primarily supported by grants from the National Institutes of Health (NIH) to V.G.: NIH Director's New Innovator DP2NS087949 and PECASE, NIH BRAIN Armamentarium UF1MH128336, NIH Pioneer DP1NS111369 and SPARC OT2OD024899. Additional funding includes the Vallee Foundation (V.G.), the Moore Foundation (V.G.), the CZI Neurodegeneration Challenge Network (V.G. and C.E.), the NSF NeuroNex Technology Hub grant 1707316 (V.G.), the Heritage Medical Research Institute (V.G.) and the Beckman Institute for CLARITY, Optogenetics, and Vector Engineering Research (CLOVER) for technology development and dissemination (V.G.), NIH BRAIN UG3MH120095 (J.T.T. and V.G.), CNPRC base grant (NIH P51 OD011107, A.S.F.), the Swiss National Science Foundation (310030_188952, A.K), the Synapsis Foundation (grant 2019-PI02, A.K.), and the Swiss Multiple Sclerosis Society (A.K.). C.E. is an investigator at the Howard Hughes Medical Institute. Diagrams in the manuscript were created with BioRender.com. Contributions. X.C. and V.G. designed the experiments. X.C., D.A.W., J. T.T., M. Z., D.S.B., H.S., S.R.K., T.F.S., E.E.S., D.G., V.N. performed experiments. D.A.W. assisted with the characterization of the vectors across models. D.S.B. and C.E. designed and conducted the in vivo Hevin expression experiments. J.T.T., M.Z., D.G., V.O., N.T., N.W., J.M., Y.B., D.M., B.G., B.P.L., and E.S.L assisted with the characterization of variants in ex vivo macaque and human brain slices. T.M. and E.S. assisted with the SPR experiment and the Ly6a experiment in HEK293T cells. S.H. and A.K. assisted with the characterization of the vectors in pericyte-deficient mice and vascular cell type-specific transduction. S.R.K. assisted with the HBMEC experiment and early iteration of the library. C.M.A. assisted with tissue processing in the macaque. X.D. generated the AAV1 and AAV9 models. V.P. and A.A. assisted with the characterization in rat with the support of the staff at SWC. V.N. and C.T.M. assisted with the characterization of the virus in marmoset with the support of vet staff at UCSD. L.J.C. and A.F. assisted with the characterization of the virus in rhesus macaque with the support of vet staff at UC Davis. X.C. prepared the figures with input from all authors. X.C. and V.G. wrote the manuscript with input from all authors. V.G. supervised all aspects of the work. Data availability. All sequences of AAV variants developed in this study are provided in materials and methods. Key vector plasmid AAV-X1.1 is available at Addgene (#196836) and also at the Beckman Institute CLOVER Center (https://clover.caltech.edu/). Source data are provided with this paper. Other data that support the findings of this study are available from the corresponding author upon request. Code availability. The code used for M-CREATE data analysis were published previously and are available on GitHub: https://github.com/GradinaruLab/mCREATE. Competing interests. The California Institute of Technology has one patent pending for the sequences described in this manuscript, with X.C. and V.G. listed as inventors (PCT Patent Application No: PCT/US2022/027516). V.G. is a co-founder and board member of Capsida Biotherapeutics, a fully integrated AAV engineering and gene therapy company. The remaining authors declare no competing interests.

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Published - s41467-023-38582-7.pdf

Supplemental Material - 41467_2023_38582_MOESM1_ESM.pdf

Supplemental Material - 41467_2023_38582_MOESM3_ESM.pdf

Supplemental Material - 41467_2023_38582_MOESM4_ESM.mp4


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August 22, 2023
December 22, 2023