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Published December 9, 2021 | Supplemental Material
Journal Article Open

AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset


Genetic intervention is increasingly being explored as a therapeutic option for debilitating disorders of the central nervous system. The safety and efficacy of gene therapies rely upon expressing a transgene in affected cells while minimizing off-target expression. Here we show organ-specific targeting of adeno-associated virus (AAV) capsids after intravenous delivery, which we achieved by employing a Cre-transgenic-based screening platform and sequential engineering of AAV-PHP.eB between the surface-exposed AA452 and AA460 of VP3. From this selection, we identified capsid variants that were enriched in the brain and targeted away from the liver in C57BL/6J mice. This tropism extends to marmoset (Callithrix jacchus), enabling robust, non-invasive gene delivery to the marmoset brain after intravenous administration. Notably, the capsids identified result in distinct transgene expression profiles within the brain, with one exhibiting high specificity to neurons. The ability to cross the blood–brain barrier with neuronal specificity in rodents and non-human primates enables new avenues for basic research and therapeutic possibilities unattainable with naturally occurring serotypes.

Additional Information

© 2021 Nature Publishing Group. Received 16 June 2020. Accepted 22 October 2021. Published 09 December 2021. We wish to thank the entire Gradinaru laboratory for helpful discussions. We thank B. Deverman and S. Kumar for helpful discussions on M-CREATE design and implementation, as described in Kumar et al.33. We thank M. Borsos, A. Hori, T. Yan, X. Qu and Y. Chen for their technical assistance. We are grateful to I. Antoshechkin and the Millar and Muriel Jacobs Genetics and Genomics Core at the California Institute of Technology for assistance with next-generation sequencing. We thank M. Smith for helpful discussions and assistance with the bioinformatics pipeline. This work was primarily supported by Defense Advanced Research Projects Agency grant W911NF-17-2-0036 to V.G. and grants from the National Institutes of Health (NIH) to V.G.: Director's New Innovator and PECASE DP2NS087949, BRAIN R01MH117069, BRAIN U01 UMH128336A and NIH Pioneer DP1OD025535. D.G was supported by the National Sciences and Engineering Research Council of Canada. Additional support for revision experiments was provided by K. L. Yang and the Hock E. Tan Center for Molecular Therapeutics in Neuroscience at MIT to G.F., the James and Patricia Poitras Center for Psychiatric Disorders Research at MIT to G.F., NIH grant U24 OD026638 to G.F., Guangdong Provincial Fund for Basic and Applied Basic Research (2019B1515130004) to Y.C., the Shenzhen Knowledge Innovation Program (JCYJ20170413165053031) to Y.C., the National Key R&D Program of China (2018YFE0203600) to Y.C., the Areas of Excellence Scheme of the University Grants Committee of Hong Kong (AoE/M-604/16) to N.Y.I. and the Hong Kong Center for Neurodegenerative Diseases (INNOHK18SC01) to N.Y.I. In addition, this work is funded by the Beckman Institute at the California Institute of Technology, the Vallee Foundation and the Moore Foundation. V.G. is a Heritage Principal Investigator supported by the Heritage Medical Research Institute. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. These authors contributed equally: David Goertsen, Nicholas C. Flytzanis, Nick Goeden. Contributions. D.G., N.C.F and N.G. analyzed all data and prepared all figures, with input from V.G. N.C.F and N.G. designed and performed the variant selection experiments, characterized the variants in mice and prepared the virus for the pooled marmoset experiments. J.P and A.C. performed National Institutes of Health (NIH) marmoset experiments and analyzed the associated data. J.P. supervised the NIH experiments and helped prepare the associated figure. During revision, the Massachusetts Institute of Technology (MIT), the Shenzhen Institute of Advanced Technology (SIAT) and the Hong Kong University of Science and Technology (HKUST) provided additional marmoset data on controls (AAV-PHP.eB) and to increase 'n' for all cohorts. D.G. and M.C. prepared the virus for single-variant characterization and performed marmoset single-variant characterization tissue analysis of MIT and NIH marmoset tissues. Q.Z. and J.S. assisted with the MIT AAV injections, animal perfusion and tissue sample collection, under the supervision of G.F. Yijing Chen and Y.F. performed single-variant characterization of the SIAT animals and analyzed the associated data, under the supervision of Yu Chen. L.W. supervised the marmoset breeding at the SIAT. Y.D. coordinated with D.G. for experiments at the SIAT, under the supervision of N.Y.I. N.C.F., N.G., D.G. and V.G. wrote the manuscript, with input from all authors. No experimental work was performed during the Los Angeles COVID-19 stay-at-home order. V.G. supervised all aspects of the work. Data availability. The NGS datasets for capsid selection and marmoset pooled screening that are reported in this article are available under Sequence Read Archive accession code PRJNA769435. The following vector plasmids were deposited in Addgene for distribution, and viruses might be available for commonly packaged genomes (http://www.addgene.org): AAV.CAP-B10 (Addgene, 175004) and AAV.CAP-B22 (Addgene, 175005). All other constructs and tools will be available through the Beckman Institute CLOVER Center (https://clover.caltech.edu/). The data that support the findings of this study are available from the corresponding author upon reasonable request. Code availability. The codes 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 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. 16/582,635, 2020). V.G. is a co-founder and board member and N.C.F. and N.G. are co-founders and employees of Capsida Biotherapeutics, a fully integrated AAV engineering and gene therapy company. Capsida Biotherapeutics did not provide funding and had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The remaining authors declare no competing interests. Peer review information. Nature Neuroscience thanks Subhojit Roy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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