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

Creators: Chen, Xinhong; Wolfe, Damien A.; Sivadasan Bindu, Dhanesh; Zhang, Mengying; Taskin, Naz; Goertsen, David; Shay, Timothy F.; Sullivan, Erin E.; Huang, Sheng-Fu; Ravindra Kumar, Sripriya; Arokiaraj, Cynthia M.; Plattner, Viktor M.; Campos, Lillian J.; Mich, John; Monet, Deja; Ngo, Victoria; Ding, Xiaozhe; Omstead, Victoria; Weed, Natalie; Bishaw, Yeme; Gore, Bryan; Lein, Ed S.; Akrami, Athena; Miller, Cory; Levi, Boaz P.; Keller, Annika; Ting, Jonathan T.; Fox, Andrew S.; Eroglu, Cagla; Gradinaru, Viviana

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Abstract

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 rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), andex vivohuman 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. Vasculature-secreted Hevin (a synaptogenic protein) rescued synaptic deficits in a mouse model.

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 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, Min Jee Jang and Cameron Jackson for attempt on FISH 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 service. 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 1UF1MH128336-01, NIH Pioneer 5DP1NS111369-04 and SPARC 1OT2OD024899. Additional funding includes the Vallee Foundation (V.G.), the Moore Foundation (V.G.), the CZI Neurodegeneration Challenge Network (V.G. and C.E.), and 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, V.G.), CNPRC base grant (NIH P51 OD011107, A.F.), the Swiss National Science Foundation (310030_188952, A.K), the Synapsis (grant 2019-PI02, A.K.), the Swiss Multiple Sclerosis Society (A.K.). C.E. is an investigator of the Howard Hughes Medical Institute. 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.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 macaque. X.Z generated the AAV1 and AAV9 models. V.P. and A.K. 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. Competing Interest Statement. The California Institute of Technology has filed patent applications for the work described in this manuscript, with X.C. 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.

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