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Published October 2023 | Published
Journal Article Open

Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain


Crossing the blood–brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood–brain barrier in non-human primates. Here we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques, which has improved delivery efficiency in the brains of multiple non-human primate species: marmoset, rhesus macaque and green monkey. CAP-Mac is neuron biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques and is vasculature biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labelling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of non-human primates.

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 wish to thank the entire Gradinaru laboratory, the Beckman Institute CLOVER Center staff and members of Capsida Biotherapeutics for helpful discussions. We thank D. Lidgate for her helpful input in planning and writing the paper. We thank C. Oikonomou for help with manuscript editing, M. Borsos for his assistance in mouse pup injection and A. Kahan and G. M. Coughlin for their helpful discussion and input in planning rhesus macaque experiments and experimental cargo. We also thank Nationwide Histology Incorporated for their assistance in providing assessments on degeneration and inflammation pathology. J.C.O., K.M.R., J.S., J.W., R.J.B., N.C.F. and N.G. (Capsida Biotherapeutics) would like to thank M. Weed and the whole team at Virscio for their help with the design and execution of green monkey experiments. We are grateful to I. Antoshechkin and the Millard and Muriel Jacobs Genetics and Genomics Core at the California Institute of Technology for assistance with NGS. We are grateful to the research and veterinarian staff at the California National Primate Research Center (CNPRC) for their aid with studies involving rhesus macaques. This work was funded by grants from the National Institutes of Health (NIH): NIH Pioneer DP1NS111369 (to V.G.); P51OD011107 (to the California National Primate Research Center), R01HD091325 (to L.T.); U19NS123719 (to L.T.); UG3MH120095 (to J.T.T. and B.P.L.); P51OD010425 (to the Washington National Primate Research Center); U42OD011123 (to the Washington National Primate Research Center); BRAIN Armamentarium UF1MH128336 (to V.G., T.F.S., L.T. and A.S.F.), and in part by Aligning Science Across Parkinson's (ASAP-020495 to V.G., A.S.F. and L.T.) through the Michael J. Fox Foundation for Parkinson's Research (MJFF). The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the aforementioned funding agencies. For the purpose of open access, the authors have applied a CC-BY public copyright license to all Author Accepted Manuscripts arising from this submission. Figures were created using images from BioRender.com and were obtained with a publication license. Data availability: The capsid plasmid used to produce AAV.CAP-Mac is available on Addgene (200658; RRID: Addgene_200658). Imaging datasets are available on the Brain Imaging Library (https://doi.org/10.35077/g.948). Source data are available for Figs. 2, 3, 5 and 6 and Extended Data Figs. 2, 3, 6 and 7. All other data that support the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper. Code availability: NeuN quantification was performed using CellPose (https://www.cellpose.org/; RRID: SCR_021716)73 and ImageJ (RRID: SCR_003070). All NGS data were processed using previously published open-source software from our lab (https://github.com/GradinaruLab/protfarm and https://github.com/GradinaruLab/mCREATE)16. All code specifically associated with this manuscript is available via GitHub at https://github.com/GradinaruLab/CAP-Mac (https://doi.org/10.5281/zenodo.8040857). Contributions: These authors contributed equally: Miguel R. Chuapoco, Nicholas C. Flytzanis, Nick Goeden. M.R.C. and N.C.F. wrote the manuscript, with input from all authors. M.R.C. designed, performed and analysed the data for the rhesus macaque and rodent experiments and prepared all the figures. N.C.F., K.Y.C. and B.E.D. designed; N.C.F. and K.Y.C. performed; and N.C.F. analysed the associated data for the viral library screening experiments in common marmosets. N.C.F. and N.G. designed, performed and analysed the data of the pooled testing experiments in rhesus macaques. N.C.F. and N.G. designed the green monkey experiments; N.C.F., N.G., J.C.O. and K.M.R. performed these experiments; and J.C.O. and K.M.R. analysed the associated data and helped prepare the associated figures. J.S., J.W. and R.J.B. designed and performed the human neuron experiments, analysed the associated data and prepared the associated figures. L.J.C. designed and performed the rhesus macaque experiments. C.M.A. performed the rhesus macaque spinal cord and dorsal root ganglia analysis and imaging. K.N.M.M. and J.S. performed the ex vivo macaque two-photon imaging and analysis. X.C. made the virus and planned and analysed the ex vivo adult macaque tissue characterization. A.L. and V.P.S. handled and processed the adult common marmoset tissue for individual characterization. T.F.S. analysed the second-round viral library screening experiment in the common marmoset and generated the sequence clustering graphs. M.J.J. helped with the imaging analysis. J.V. helped perform the rhesus macaque neuron tracing. J.M., Y.B. and B.G. performed and analysed the quantitative PCR for the ex vivo adult macaque tissue characterization. V.O., N.T. and N.W. performed the adult macaque ex vivo brain slice culture, immunohistochemistry and imaging. J.T.T. and B.P.L. planned and supervised all the aspects of the ex vivo adult macaque slice experiments. C.T.M. supervised the common marmoset individual characterizations and J.P. supervised the common marmoset selections. L.T. designed and supervised the ex vivo macaque two-photon imaging. A.S.F. designed, performed and supervised the rhesus macaque experiments. N.C.F. supervised all the aspects of the green monkey and iPSC work. V.G. supervised all the aspects of the library screening, pooled testing and rhesus macaque work, and contributed to the associated experimental design, data analysis and manuscript writing. Competing 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. A license for US patent application no. PCT/US21/46904 has been issued to Capsida Biotherapeutics. B.D. is a scientific founder and scientific advisor of Apertura Gene Therapy and is on the scientific advisory board of Tevard Biosciences. B.P.L. is on the scientific advisory board of Patch Biosciences. The remaining authors declare no competing interests.

Attached Files

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November 7, 2023
January 9, 2024