Primate-conserved Carbonic Anhydrase IV and murine-restricted Ly6c1 are new targets for crossing the blood-brain barrier
Abstract
The blood-brain barrier (BBB) presents a major challenge to delivering large molecules to study and treat the central nervous system (CNS). This is due in part to the scarcity of effective targets for BBB crossing, the identification of which is the crucial first step of drug development. Here, we leveraged a panel of adeno-associated viruses (AAVs) previously identified through directed evolution for improved BBB transport to reverse engineer protein targets for enhanced BBB crossing. We identify both murine-restricted Ly6c1 and primate-conserved carbonic anhydrase IV (Car4; CA4) as novel receptors for crossing the BBB. We demonstrate how these receptors can unlock new experimental and computational target-focused engineering strategies by creating the enhanced Ly6c1-binding vector AAV-PHP.eC and by applying AlphaFold2-enabled in silico methods to rank capsids against identified receptors and generate capsid-receptor binding models. Here, with Car4, we add a completely new receptor to the very short list currently available for crossing the BBB in humans and, with Ly6c1, we validate a pipeline for receptor-targeted engineering. The identification of Car4/CA4 and structural insights from computational modeling provide new paths toward human brain-penetrant chemicals (drugs) and biologicals (including gene delivery).
Additional Information
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. We thank Catherine Oikonomou for help with manuscript editing, Michael Altermatt for assisting in filtering scRNAseq datasets for membrane proteins, Min Jee Jang for designing RNA sequencing variant barcodes, Damien A. Wolfe for assisting in mouse perfusion and tissue collection, Josette Medicielo for plasmid purification, and Julie Miwa (Lehigh) for sharing a lynx1 expression plasmid. We thank the entire Gradinaru lab and CLOVER center staff for helpful discussion. Figures were created using images from BioRender.com. This work was primarily supported by NIH PIONEER DP1OD025535 (to V.G.) and the Beckman Institute for CLARITY, Optogenetics & 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. and E.E.S. produced AAVs. T.F.S. and J.V. performed SPR experiments. T.F.S. and D.B. analyzed the scRNAseq 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 and tested AAVs in wild type mice. T.F.S. and V.G. supervised and V.G. funded the project. Competing Interest Statement. The California Institute of Technology has filed a provisional patent for this work with T.F.S., X.D., and V.G. listed as inventors. 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.Attached Files
Submitted - 2023.01.12.523632v1.full.pdf
Supplemental Material - media-1.pdf
Files
| Name | Size | Download all |
|---|---|---|
|
md5:62492be9dc927f8a1d02da9ccf55c4cd
|
2.0 MB | Preview Download |
|
md5:a5ac1a0fb63a441b1435c2de0ec4f57f
|
1.1 MB | Preview Download |
Additional details
- Eprint ID
- 120155
- Resolver ID
- CaltechAUTHORS:20230316-182564000.42
- DP1OD025535
- NIH
- Beckman Institute for CLARITY, Optogenetics and Vector Engineering Research
- Created
-
2023-03-18Created from EPrint's datestamp field
- Updated
-
2023-03-18Created from EPrint's last_modified field
- Caltech groups
- Division of Biology and Biological Engineering