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Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing

Brown, David and Altermatt, Michael and Dobreva, Tatyana and Chen, Sisi and Wang, Alexander and Thomson, Matt and Gradinaru, Viviana (2021) Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20210629-153450418

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Abstract

Engineered variants of recombinant adeno-associated viruses (rAAVs) are being developed rapidly to meet the need for gene-therapy delivery vehicles with particular cell-type and tissue tropisms. While high-throughput AAV engineering and selection methods have generated numerous variants, subsequent tropism and response characterization have remained low throughput and lack resolution across the many relevant cell and tissue types. To fully leverage the output of these large screening paradigms across multiple targets, we have developed an experimental and computational single-cell RNA sequencing (scRNA-seq) pipeline for in vivo characterization of barcoded rAAV pools at unprecedented resolution. Using our platform, we have corroborated previously reported viral tropisms and discovered unidentified AAV capsid targeting biases. As expected, we observed that the tropism profile of AAV.CAP-B10 in mice was shifted toward neurons and away from astrocytes when compared with AAV-PHP.eB. Our transcriptomic analysis revealed that this neuronal bias is mainly due to increased targeting efficiency for glutamatergic neurons, which we confirmed by RNA fluorescence in situ hybridization. We further uncovered cell subtype tropisms of AAV variants in vascular and glial cells, such as low transduction of pericytes and Myoc+ astrocytes. Additionally, we have observed cell-type-specific responses to systemic AAV-PHP.eB administration, such as upregulation of genes involved in p53 signaling in endothelial cells three days post-injection, which return to control levels by day twenty-five. Such ability to parallelize the characterization of AAV tropism and simultaneously measure the transcriptional response of transduction will facilitate the advancement of safe and precise gene delivery vehicles.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/2021.06.25.449955DOIDiscussion Paper
ORCID:
AuthorORCID
Brown, David0000-0002-9757-1744
Altermatt, Michael0000-0003-2841-5374
Dobreva, Tatyana0000-0002-2625-8873
Chen, Sisi0000-0001-9448-9713
Gradinaru, Viviana0000-0001-5868-348X
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. Version 1 - June 25, 2021; Version 2 - July 10, 2021. We thank the Gradinaru and Thomson labs for helpful discussions, Allan-Hermann Pool for advice on the mouse brain tissue dissociation procedure, Jeff Park for advice on 10X Genomics Chromium single-cell library preparation, Min Jee Jang for help in designing probes and troubleshooting FISH-HCR, and Ben Deverman and Ken Chan for early discussions on strategy. This work was supported by the NIH Pioneer DP1OD025535, Beckman Institute for CLARITY, Optogenetics and Vector Engineering Research at Caltech, the Single-Cell Profiling and Engineering Center (SPEC) in the Beckman Institute at Caltech, and the Curci Foundation. V.G. and M.T. are Heritage Principal Investigators supported by the Heritage Medical Research Institute. Author contributions: D.B., M.A., T.D., and V.G. conceived the project and designed the experiments. S.C. and M.T. provided critical single-cell RNA sequencing expertise. T.D., M.A., and D.B. prepared the DNA constructs and produced virus. M.A. performed the injections, tissue dissociation, histology, imaging and image quantification. D.B. and T.D. performed the single-cell library preparation and prepared samples for sequencing. D.B. and M.A. built the data processing pipeline. D.B., M.A., T.D., and A.W. performed the analysis. All authors contributed to the MS as drafted by D.B., M.A., and V.G. M.T. supervised single-cell RNA sequencing computational pipelines while V.G. supervised the overall project. The authors have declared no competing interest.
Group:Heritage Medical Research Institute
Funders:
Funding AgencyGrant Number
NIHDP1OD025535
Caltech Beckman InstituteUNSPECIFIED
Shurl and Kay Curci FoundationUNSPECIFIED
Heritage Medical Research InstituteUNSPECIFIED
Record Number:CaltechAUTHORS:20210629-153450418
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210629-153450418
Official Citation:Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing. David Brown, Michael Altermatt, Tatyana Dobreva, Sisi Chen, Alexander Wang, Matt Thomson, Viviana Gradinaru. bioRxiv 2021.06.25.449955; doi: https://doi.org/10.1101/2021.06.25.449955
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:109639
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:29 Jun 2021 16:11
Last Modified:12 Jul 2021 22:49

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