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Published April 21, 2023 | Published + Supplemental Material
Journal Article Open

Primate-conserved carbonic anhydrase IV and murine-restricted LY6C1 enable blood-brain barrier crossing by engineered viral vectors


The blood-brain barrier (BBB) presents a major challenge for delivering large molecules to study and treat the central nervous system. This is due in part to the scarcity of targets known to mediate BBB crossing. To identify novel targets, we leverage a panel of adeno-associated viruses (AAVs) previously identified through mechanism-agnostic directed evolution for improved BBB transcytosis. Screening potential cognate receptors for enhanced BBB crossing, we identify two targets: murine-restricted LY6C1 and widely conserved carbonic anhydrase IV (CA-IV). We apply AlphaFold-based in silico methods to generate capsid-receptor binding models to predict the affinity of AAVs for these identified receptors. Demonstrating how these tools can unlock target-focused engineering strategies, we create an enhanced LY6C1-binding vector, AAV-PHP.eC, that, unlike our prior PHP.eB, also works in Ly6a-deficient mouse strains such as BALB/cJ. Combined with structural insights from computational modeling, the identification of primate-conserved CA-IV enables the design of more specific and potent human brain–penetrant chemicals and biologicals, including gene delivery vectors.

Additional Information

© 2023 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY). We thank C. Oikonomou for help with manuscript editing, M. Altermatt for assisting in filtering single-cell RNA sequencing datasets for membrane proteins, M. J. Jang for designing RNA sequencing variant barcodes, J. Medicielo for plasmid purification, and J. Miwa (Lehigh) for sharing a lynx1 expression plasmid. We thank the Caltech Protein Expression Center supported by the Beckman Institute for SPR studies, protein expression, and purification. We thank A. D. Steele (CPP) for contributions to the Caltech - CPP ASPIRE Program and the entire Gradinaru laboratory and the Beckman Institute CLOVER Center staff for helpful discussions. Figures were created using images from BioRender.com. This work was primarily supported by the NIH PIONEER DP1OD025535 (to V.G.), NIH BRAIN Initiative Armamentarium UF1MH128336 (to V.G. and T.F.S.) and U24MH131054 (to T.F.S. and V.G.), and the Beckman Institute for CLARITY, Optogenetics and Vector Engineering Research (CLOVER) for technology development and dissemination (to T.F.S. and V.G.). Author contributions: T.F.S. and V.G. conceived the project. T.F.S. and V.G. wrote the manuscript and prepared figures with input from all authors. X.D. and A.W.L. optimized LY6A-Fc expression protocol, and A.W.L. produced LY6A-Fc protein. T.F.S., E.E.S., and D.A.W. produced AAVs. T.F.S. and J.V. performed SPR experiments. T.F.S. and D.B. analyzed the single-cell RNA sequencing dataset. T.F.S. and E.E.S. developed and E.E.S. performed the cell culture infectivity assay. D.G. developed and implemented the in vitro transduction quantification and plotting pipeline, performed data analysis, and, with T.F.S., prepared in vitro transduction quantification plots. E.E.S. and M.B. performed immunofluorescence experiments. E.E.S., X.C., and M.B. performed Car4-KO experiments, X.D. performed APPRAISE-AAV and developed computational structural modeling strategies. T.F.S., S.R.K., E.E.S., and X.C. performed and analyzed AAV-PHP.eC selections. E.E.S., X.C., A.C., and D.A.W. tested AAVs in WT mice. T.F.S. and V.G. supervised and V.G. funded the project. Data and materials availability: The NGS datasets that are reported here have been made available with SRA accession code PRJNA942970. Single-cell RNA sequencing datasets are available at CaltechDATA (data.caltech.edu/records/2090). Plasmids or viruses not available at Addgene may be requested from the Caltech CLOVER Center (clover.caltech.edu). The AAV-PHP.eC sequence is deposited in GenBank with accession code OQ606833. All in vitro transduction image processing was performed using our custom Python image processing pipeline. An archive of the image processing pipeline used for this study is available at Zenodo (https://doi.org/10.5281/zenodo.7697435), and future updates of the pipeline can be found on GitHub (https://github.com/GradinaruLab/in-vitro-transduction-assay). The code used for M-CREATE data analyses (22) is available on GitHub (https://github.com/GradinaruLab/mCREATE). The code used for single-cell RNA sequencing analyses (48) is available through GitHub (https://github.com/GradinaruLab/aavomics). The code used for APPRAISE-AAV (45) is available on GitHub (https://github.com/GradinaruLab/APPRAISE) and through a web-based notebook (https://tiny.cc/APPRAISE). All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Competing interests: The California Institute of Technology has filed a patent for this work with T.F.S., X.D., and V.G. listed as inventors. V.G. is a cofounder and board member of Capsida Biotherapeutics, a fully integrated AAV engineering and gene therapy company. The other authors declare that they have no competing interests.

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August 22, 2023
December 22, 2023