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Systemic AAV vectors for widespread and targeted gene delivery in rodents

A Publisher Correction to this article was published on 16 July 2019

This article has been updated

Abstract

We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.

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Fig. 1: Overview of the protocol.
Fig. 2: AAV-PHP.eB and gene regulatory elements enable cell type–specific gene expression in the brain.
Fig. 3: AAV-PHP.S transduces neurons throughout the PNS.
Fig. 4: AAV-PHP.S for mapping the anatomy and physiology of the heart.
Fig. 5: AAV-PHP.B and AAV-PHP.eB can be used in several mouse and rat strains.
Fig. 6: A modular AAV toolbox for cell type–specific gene expression.
Fig. 7: Time line and AAV harvest procedure.
Fig. 8: AAV purification procedure.

Change history

  • 16 July 2019

    During the production process, the authors of this paper supplied revised versions of Figs. 2–5, Supplementary Tables 1–4, and Supplementary Videos 1–3, but because of publisher error, these revised items were not included in the final published version of the protocol. The figures have been updated in the PDF and HTML versions of the paper, and the revised Supplementary Information files are now available online. We note that the figures have been revised to improve their resolution only; the content of the figures and the data reflected remain unchanged. Also, print requirements impose some limits on figure resolution, but the authors have made very high-resolution versions of Figs. 2–5 available at as Source data.

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Acknowledgements

We thank M. Fabiszak (W. Freiwald lab, Rockefeller University) and N.C. Flytzanis (V. Gradinaru lab) for the images in Fig. 5d and e, respectively. We also thank M.S. Ladinsky at the Biological and Cryogenic Transmission Electron Microscopy Center (California Institute of Technology (Caltech)) for preparing transmission electron microscopy samples and for acquiring the image shown in Fig. 7b. We are grateful to Y. Lei for help with cloning and K. Lencioni for performing tail-vein injections in rats. The images in Fig. 5a,b were acquired in the Biological Imaging Facility, with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. AAV-PHP capsids are dedicated to the memory of Paul H. Patterson (P.H.P.), who passed away during the preparation of the manuscript describing AAV-PHP.B[3]. This work was primarily supported by the National Institutes of Health (NIH) through grants to V.G.: Director’s New Innovator grant DP2NS087949 and PECASE; National Institute on Aging grant R01AG047664; BRAIN grant U01NS090577; SPARC grant OT2OD023848-01 (to V.G. and K.S.); and the Defense Advanced Research Projects Agency (DARPA) Biological Technologies Office (BTO). Additional funding included the NSF NeuroNex Technology Hub grant 1707316, and funds from the Curci Foundation, the Beckman Institute, and the Rosen Center at Caltech. V.G. is a Heritage Principal Investigator supported by the Heritage Medical Research Institute. R.C.C. was supported by an American Heart Association Postdoctoral Fellowship (17POST33410404). C.C. was funded by the National Institute on Aging (F32AG054101), and P.S.R. was funded by the National Heart, Lung, and Blood Institute (F31HL127974).

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Contributions

R.C.C. and V.G. wrote the manuscript with input from all coauthors. S.R.K., K.Y.C., K.B., and B.E.D. optimized the viral production and purification protocols. R.C.C., S.R.K., K.Y.C., C.C., H.K., P.S.R., J.D.T., K.S., B.E.D., and V.G. designed and performed the experiments, analyzed the data, and prepared the figures. M.J.J. analyzed the data in Fig. 2c. V.G. supervised all aspects of the project. All authors edited and approved the manuscript.

Corresponding author

Correspondence to Viviana Gradinaru.

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Competing interests

The California Institute of Technology has filed patent applications related to (but not on) this work: Recombinant AAV Capsid Protein (US patent no. 9,585,971); Selective Recovery (US patent application no. 15/422,259); Targeting Peptides for Directing Adeno-Associated Viruses (AAVs) (US patent application no. 15/374,596). The authors declare no other competing interests.

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Related links

Key references using this protocol

Deverman, B. E. et al. Nat. Biotechnol. 34, 204–209 (2016): https://doi.org/10.1038/nbt.3440

Chan, K. Y. et al. Nat. Neurosci. 20, 1172–1179 (2017): https://doi.org/10.1038/nn.4593

Supplementary information

Supplementary Tables 1 and 3

Supplementary Table 2

Transfection calculator

Supplementary Table 4

Titration calculator

Supplementary Video 1

Steps 16A and 18: Pouring the density gradients and loading the virus. In Step 16A, use a 2-ml serological pipette to pour the gradients. Next, load the virus (also shown in Step 16B (Supplementary Video 2))

Supplementary Video 2

Steps 16B and 18: Pouring the density gradients and loading the virus. In Step 16B, use a 5-ml serological pipette to pour the gradients. Next, load the virus (also shown in Step16A (Supplementary Video 1))

Supplementary Video 3

Steps 26–27: Collecting the virus

Source Data, Figures 2-5

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Challis, R.C., Ravindra Kumar, S., Chan, K.Y. et al. Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nat Protoc 14, 379–414 (2019). https://doi.org/10.1038/s41596-018-0097-3

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