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Systemic AAV vectors for widespread and targeted gene delivery in rodents

Challis, Rosemary C. and Ravindra Kumar, Sripriya and Chan, Ken Y. and Challis, Collin and Beadle, Keith and Jang, Min J. and Kim, Hyun Min and Rajendran, Pradeep S. and Tompkins, John D. and Shivkumar, Kalyanam and Deverman, Benjamin E. and Gradinaru, Viviana (2019) Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nature Protocols, 14 (2). pp. 379-414. ISSN 1754-2189.

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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.

Item Type:Article
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URLURL TypeDescription ReadCube access Paper Correction ReadCube access - Publisher Correction
Challis, Rosemary C.0000-0003-3086-6553
Ravindra Kumar, Sripriya0000-0001-6033-7631
Chan, Ken Y.0000-0002-8853-5186
Challis, Collin0000-0003-4716-6086
Jang, Min J.0000-0002-1536-7177
Rajendran, Pradeep S.0000-0001-9790-0408
Shivkumar, Kalyanam0000-0002-4121-1766
Deverman, Benjamin E.0000-0002-6223-9303
Gradinaru, Viviana0000-0001-5868-348X
Alternate Title:Widespread and targeted gene expression by systemic AAV vectors: Production, purification, and administration
Additional Information:© 2019 Springer Nature Publishing AG. Published 09 January 2019. 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). Author 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. 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.
Errata: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 as Source data.
Group:Heritage Medical Research Institute, Rosen Bioengineering Center
Funding AgencyGrant Number
Caltech Beckman InstituteUNSPECIFIED
Arnold and Mabel Beckman FoundationUNSPECIFIED
Defense Advanced Research Projects Agency (DARPA)UNSPECIFIED
Curci FoundationUNSPECIFIED
Donna and Benjamin M. Rosen Bioengineering CenterUNSPECIFIED
Heritage Medical Research InstituteUNSPECIFIED
American Heart Association17POST33410404
NIH Postdoctoral FellowshipF32AG054101
NIH Postdoctoral FellowshipF31HL127974
Issue or Number:2
Record Number:CaltechAUTHORS:20180816-125507808
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:88862
Deposited By: George Porter
Deposited On:17 Aug 2018 19:35
Last Modified:09 Mar 2020 13:18

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