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Use of high-content imaging to quantify transduction of AAV-PHP viruses in the brain following systemic delivery

Smith, Edward J. and Farshim, Pamela P. and Flomen, Rachel and Jones, Samuel T. and McAteer, Sean J. and Deverman, Benjamin E. and Gradinaru, Viviana and Bates, Gillian P. (2021) Use of high-content imaging to quantify transduction of AAV-PHP viruses in the brain following systemic delivery. Brain Communications, 3 (2). Art. No. fcab105. ISSN 2632-1297. PMCID PMC8200048. doi:10.1093/braincomms/fcab105. https://resolver.caltech.edu/CaltechAUTHORS:20210629-201655221

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Abstract

The engineering of the AAV-PHP capsids was an important development for CNS research and the modulation of gene expression in the brain. They cross the blood brain barrier and transduce brain cells after intravenous systemic delivery, a property dependent on the genotype of Ly6a, the AAV-PHP capsid receptor. It is important to determine the transduction efficiency of a given viral preparation, as well as the comparative tropism for different brain cells; however, manual estimation of adeno-associated viral transduction efficiencies can be biased and time consuming. Therefore, we have used the Opera Phenix high-content screening system, equipped with the Harmony processing and analysis software, to reduce bias and develop an automated approach to determining transduction efficiency in the mouse brain. We used R Studio and ‘gatepoints’ to segment the data captured from coronal brain sections into brain regions of interest. C57BL/6J and CBA/Ca mice were injected with an AAV-PHP.B virus containing a green fluorescent protein reporter with a nuclear localization signal. Coronal sections at 600 μm intervals throughout the entire brain were stained with Hoechst dye, combined with immunofluorescence to NeuN and green fluorescent protein to identify all cell nuclei, neurons and transduced cells, respectively. Automated data analysis was applied to give an estimate of neuronal percentages and transduction efficiencies throughout the entire brain as well as for the cortex, striatum and hippocampus. The data from each coronal section from a given mouse were highly comparable. The percentage of neurons in the C57BL/6J and CBA/Ca brains was approximately 40% and this was higher in the cortex than striatum and hippocampus. The systemic injection of AAV-PHP.B resulted in similar transduction rates across the entire brain for C57BL/6J mice. Approximately 10–15% of all cells were transduced, with neuronal transduction efficiencies ranging from 5% to 15%, estimates that were similar across brain regions, and were in contrast to the much more localized transduction efficiencies achieved through intracerebral injection. We confirmed that the delivery of the AAV-PHP.B viruses to the brain from the vasculature resulted in widespread transduction. Our methodology allows the rapid comparison of transduction rates between brain regions producing comparable data to more time-consuming approaches. The methodology developed here can be applied to the automated quantification of any parameter of interest that can be captured as a fluorescent signal.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1093/braincomms/fcab105DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8200048PubMed CentralArticle
ORCID:
AuthorORCID
Farshim, Pamela P.0000-0003-4816-6635
Flomen, Rachel0000-0002-6434-5804
Deverman, Benjamin E.0000-0002-6223-9303
Gradinaru, Viviana0000-0001-5868-348X
Bates, Gillian P.0000-0002-4041-6305
Additional Information:© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 21 October 2020; Revision received: 19 March 2021; Accepted: 08 April 2021; Published: 17 May 2021. We thank Arzo Iqbal and Zoe Windsor for help with viral administration, Miguel Estevez for the ssAAV-PHP.eB-CAG-NLS-GFP virus, Robin Ketteler and Christin Luft from the High-Content Biology Laboratory at UCL and James Hutt from Perkin Elmer for helpful technical discussions. This work was supported by grants from the CHDI Foundation, UK Dementia Research Institute, which receives its funding from DRI Ltd, funded by the UK Medical Research Council, Alzheimer’s Society and Alzheimer’s Research UK, the National Institutes of Health New Innovator (DP2NS087949 to V.G.) and the Beckman Institute for CLARITY, Optogenetics and Vector Engineering Research (CLOVER) for technology development and dissemination (V.G. and B.E.D.). The authors have no competing interests to declare.
Funders:
Funding AgencyGrant Number
CHDI FoundationUNSPECIFIED
Dementia Research InstituteUNSPECIFIED
DRI Ltd.UNSPECIFIED
Medical Research Council (UK)UNSPECIFIED
Alzheimer’s SocietyUNSPECIFIED
Alzheimer’s ResearchUNSPECIFIED
NIHDP2NS087949
Caltech Beckman InstituteUNSPECIFIED
Issue or Number:2
PubMed Central ID:PMC8200048
DOI:10.1093/braincomms/fcab105
Record Number:CaltechAUTHORS:20210629-201655221
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210629-201655221
Official Citation:Edward J Smith, Pamela P Farshim, Rachel Flomen, Samuel T Jones, Sean J McAteer, Benjamin E Deverman, Viviana Gradinaru, Gillian P Bates, Use of high-content imaging to quantify transduction of AAV-PHP viruses in the brain following systemic delivery, Brain Communications, Volume 3, Issue 2, 2021, fcab105, https://doi.org/10.1093/braincomms/fcab105
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:109653
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:01 Jul 2021 17:48
Last Modified:06 Jul 2021 22:04

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