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

2022Journal article Restricted access
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dc.contributor.authorLopes, Miguel M.
dc.contributor.authorPaysan, Jacques
dc.contributor.authorRino, José
dc.contributor.authorLopes, Sara M.
dc.contributor.authorPereira de Almeida, Luís
dc.contributor.authorCortes, Luísa
dc.contributor.authorNobre, Rui Jorge
dc.date.accessioned2022-12-16T16:03:39Z
dc.date.available2022-12-16T16:03:39Z
dc.date.issued2022
dc.description© The Author(s), under exclusive licence to Springer Nature Limited 2022pt_PT
dc.description.abstractRecombinant 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.pt_PT
dc.description.sponsorshipThis work was funded by the ERDF through the Regional Operational Program Center 2020, Competitiveness Factors Operational Program (COMPETE 2020) and National Funds through FCT (Foundation for Science and Technology): Imagene (PTDC/BBB-NAN/0932/2014 | POCI-01-0145-FEDER-016807), MODELPOLYQ 2.O (CENTRO-01-0145-FEDER-181258), MJDEDIT (CENTRO-01-0145-FEDER-181266), BDFORMJD (CENTRO-01-0145-FEDER-181240), CENTRO-01-0246-FEDER-000010 (Multidisciplinary Institute of Ageing in Coimbra), BrainHealth2020 projects (CENTRO-01-0145-FEDER-000008), UID/NEU/04539/2019, UIDB/04539/2020, UIDP/04539/2020, LA/P/0058/2020, PPBI (POCI-01-0145-FEDER-022122), ViraVector (CENTRO-01-0145-FEDER-022095), CortaCAGs (PTDC/NEU-NMC/0084/2014 | POCI-01-0145-FEDER-016719), SpreadSilencing (POCI-01-0145-FEDER-029716), CancelStem (POCI-01-0145-FEDER-016390), POCI-01-0145-FEDER-030737, POCI-01-0145-FEDER-032309, as well as SynSpread, ESMI and ModelPolyQ under the EU Joint Program – Neurodegenerative Disease Research (JPND), the last two co-funded by the European Union H2020 program, GA No.643417; by National Ataxia Foundation (USA), the American Portuguese Biomedical Research Fund (APBRF) and the Richard Chin and Lily Lock Machado-Joseph Disease Research Fund. MML was supported by a PhD fellowship from FCT (2021.05776.BD).pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationGene Ther. 2022 Dec;29(12):665-679pt_PT
dc.identifier.doi10.1038/s41434-022-00372-zpt_PT
dc.identifier.eissn1476-5462
dc.identifier.issn0969-7128
dc.identifier.urihttp://hdl.handle.net/10451/55435
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherSpringer Naturept_PT
dc.relationPOCI-01-0145-FEDER-016807pt_PT
dc.relationImaging Gene Therapy in the Brain
dc.relationCENTRO-01-0145-FEDER-181266pt_PT
dc.relationCENTRO-01-0145-FEDER-181240pt_PT
dc.relationCENTRO-01-0246-FEDER-000010pt_PT
dc.relationCENTRO-01-0145-FEDER-000008pt_PT
dc.relationCNC. IBILI
dc.relationCenter for Innovative Biomedicine and Biotechnology
dc.relationCenter for Innovative Biomedicine and Biotechnology
dc.relationCENTRO-01-0145-FEDER-022095pt_PT
dc.relationPOCI-01-0145-FEDER-016719pt_PT
dc.relationPOCI-01-0145-FEDER-029716pt_PT
dc.relationPOCI-01-0145-FEDER-016390pt_PT
dc.relationPOCI-01-0145-FEDER-030737pt_PT
dc.relationPOCI-01-0145-FEDER-032309pt_PT
dc.relationERA-NET for establishing synergies between the Joint Programming on Neurodegenerative Diseases Research and Horizon 2020
dc.relationEnhanced AAV vector toolkit for the generation of novel animal models and therapeutic strategies for polyglutamine diseases
dc.relation.publisherversionhttps://www.nature.com/gt/pt_PT
dc.titleA new protocol for whole-brain biodistribution analysis of AAVs by tissue clearing, light-sheet microscopy and semi-automated spatial quantificationpt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardTitleImaging Gene Therapy in the Brain
oaire.awardTitleCNC. IBILI
oaire.awardTitleCenter for Innovative Biomedicine and Biotechnology
oaire.awardTitleCenter for Innovative Biomedicine and Biotechnology
oaire.awardTitleERA-NET for establishing synergies between the Joint Programming on Neurodegenerative Diseases Research and Horizon 2020
oaire.awardTitleEnhanced AAV vector toolkit for the generation of novel animal models and therapeutic strategies for polyglutamine diseases
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/9471 - RIDTI/PTDC%2FBBB-NAN%2F0932%2F2014/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UID%2FNEU%2F04539%2F2019/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F04539%2F2020/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F04539%2F2020/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/9471 - RIDTI/PTDC%2FNEU-NMC%2F0084%2F2014/PT
oaire.awardURIinfo:eu-repo/grantAgreement/EC/H2020/643417/EU
oaire.awardURIinfo:eu-repo/grantAgreement/FCT//2021.05776.BD/PT
oaire.citation.endPage679pt_PT
oaire.citation.issue12pt_PT
oaire.citation.startPage665pt_PT
oaire.citation.titleGene Therapypt_PT
oaire.citation.volume29pt_PT
oaire.fundingStream9471 - RIDTI
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStream9471 - RIDTI
oaire.fundingStreamH2020
person.familyNameRino Henriques
person.givenNameJosé Miguel
person.identifier.ciencia-idA117-FBDB-A27B
person.identifier.orcid0000-0002-0637-4527
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100008530
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameEuropean Commission
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.rightsrestrictedAccesspt_PT
rcaap.typearticlept_PT
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