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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

Gene Therapy volume 29, pages 665–679 (2022)Cite this article

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Abstract

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.

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Fig. 1: Schematic representation of the procedure for the evaluation of the potential of rAAVs to achieve a widespread biodistribution throughout the brain after an intravenous injection.
Fig. 2: Evaluation of rAAV9 biodistribution throughout the brain following an intravenous injection.
Fig. 3: Preferential cell tropism of rAAV9 in the hippocampus and cerebellum upon intravenous administration.
Fig. 4: LSCM imaging of thick brain sections cleared and labelled with “Binaree Tissue Clearing Kit for Immunostaining” for the evaluation of rAAV9 biodistribution and tropism in the cerebellum.
Fig. 5: Tissue clearing of whole brain hemispheres with “Binaree Tissue Clearing Kit”.
Fig. 6: Spatial analysis and quantification of EGFP fluorescence at a whole-organ level with the QUINT workflow.
Fig. 7: Analysis of dendrite arborization of Purkinje cells by tissue clearing and LSFM.

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All data generated or analysed during this study are included in this published article (and its supporting information files) or are available from the corresponding author on reasonable request.

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Funding

This 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).

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Author notes

  1. These authors contributed equally: Luís Pereira de Almeida, Luísa Cortes, Rui Jorge Nobre.

Authors and Affiliations

  1. CNC – Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal

    Miguel M. Lopes, Sara M. Lopes, Luís Pereira de Almeida, Luísa Cortes & Rui Jorge Nobre

  2. CIBB – Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal

    Miguel M. Lopes, Sara M. Lopes, Luís Pereira de Almeida, Luísa Cortes & Rui Jorge Nobre

  3. IIIUC – Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal

    Miguel M. Lopes, Sara M. Lopes, Luísa Cortes & Rui Jorge Nobre

  4. Carl Zeiss Microscopy GmbH, Jena, Germany

    Jacques Paysan

  5. iMM – Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal

    José Rino

  6. ViraVector – Viral Vectors for Gene Transfer Core Facility, University of Coimbra, Coimbra, Portugal

    Luís Pereira de Almeida & Rui Jorge Nobre

  7. FFUC – Faculty of Pharmacy of the University of Coimbra, Coimbra, Portugal

    Luís Pereira de Almeida

  8. MICC-CNC – Microscopy Imaging Center of Coimbra – CNC, University of Coimbra, Coimbra, Portugal

    Luísa Cortes

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Contributions

Conceptualization, MML, LPA, LC, RJN; Methodology, MML, LC, RJN; Investigation, MML, JP, JR, SML, LC; Formal Analysis, MML, LC; Visualization, MML, SML, LC; Writing – Original Draft, MML; Writing – Review & Editing, MML, JP, JR, SML, LPA, LC, RJN; Resources, JP, JR, LPA; Funding Acquisition, LPA, LC, RJN; Supervision, LPA, LC, RJN.

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Correspondence to Luís Pereira de Almeida, Luísa Cortes or Rui Jorge Nobre.

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The authors received no specific funding from Binaree, Inc. for the development of the present work. JP works for Carl Zeiss Microscopy GmbH, ZEISS Group. The other authors declare no competing interests.

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Lopes, M.M., Paysan, J., Rino, J. et al. A new protocol for whole-brain biodistribution analysis of AAVs by tissue clearing, light-sheet microscopy and semi-automated spatial quantification. Gene Ther 29, 665–679 (2022). https://doi.org/10.1038/s41434-022-00372-z

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