A new protocol for whole-brain biodistribution analysis of AAVs by tissue clearing, light-sheet microscopy and semi-automated spatial quantification.

Autor: Lopes MM; CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.; CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal.; IIIUC - Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal., Paysan J; Carl Zeiss Microscopy GmbH, Jena, Germany., Rino J; iMM - Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal., Lopes SM; CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.; CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal.; IIIUC - Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal., Pereira de Almeida L; CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal. luispa@cnc.uc.pt.; CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal. luispa@cnc.uc.pt.; ViraVector - Viral Vectors for Gene Transfer Core Facility, University of Coimbra, Coimbra, Portugal. luispa@cnc.uc.pt.; FFUC - Faculty of Pharmacy of the University of Coimbra, Coimbra, Portugal. luispa@cnc.uc.pt., Cortes L; CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal. lcortes@cnc.uc.pt.; CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal. lcortes@cnc.uc.pt.; IIIUC - Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal. lcortes@cnc.uc.pt.; MICC-CNC - Microscopy Imaging Center of Coimbra - CNC, University of Coimbra, Coimbra, Portugal. lcortes@cnc.uc.pt., Nobre RJ; CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal. rui.nobre@cnc.uc.pt.; CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal. rui.nobre@cnc.uc.pt.; IIIUC - Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal. rui.nobre@cnc.uc.pt.; ViraVector - Viral Vectors for Gene Transfer Core Facility, University of Coimbra, Coimbra, Portugal. rui.nobre@cnc.uc.pt.
Jazyk: angličtina
Zdroj: Gene therapy [Gene Ther] 2022 Dec; Vol. 29 (12), pp. 665-679. Date of Electronic Publication: 2022 Nov 01.
DOI: 10.1038/s41434-022-00372-z
Abstrakt: Recombinant adeno-associated virus (rAAV) has become one of the most promising gene delivery systems for both in vitro and in vivo applications. However, a key challenge is the lack of suitable imaging technologies to evaluate delivery, biodistribution and tropism of rAAVs and efficiently monitor disease amelioration promoted by AAV-based therapies at a whole-organ level with single-cell resolution. Therefore, we aimed to establish a new pipeline for the biodistribution analysis of natural and new variants of AAVs at a whole-brain level by tissue clearing and light-sheet fluorescence microscopy (LSFM). To test this platform, neonatal C57BL/6 mice were intravenously injected with rAAV9 encoding EGFP and, after sacrifice, brains were processed by standard immunohistochemistry and a recently released aqueous-based clearing procedure. This clearing technique required no dedicated equipment and rendered highly cleared brains, while simultaneously preserving endogenous fluorescence. Moreover, three-dimensional imaging by LSFM allowed the quantitative analysis of EGFP at a whole-brain level, as well as the reconstruction of Purkinje cells for the retrieval of valuable morphological information inaccessible by standard immunohistochemistry. In conclusion, the pipeline herein described takes the AAVs to a new level when coupled to LSFM, proving its worth as a bioimaging tool in tropism and gene therapy studies.
(© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)
Databáze: MEDLINE
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