Spatial genomics of AAV vectors reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo
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1.
California Institute of Technology
- 2. Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA
Abstract
Cell-type-specific regulatory elements such as enhancers can direct expression of recombinant adeno-associated viruses (AAVs) to specific cell types, but this approach is limited by the relatively small packaging capacity of AAVs. In this study, we used spatial genomics to show that transcriptional crosstalk between individual AAV genomes provides a general method for cell-type-specific expression of large cargo by separating distally acting regulatory elements into a second AAV genome. We identified and profiled transcriptional crosstalk in AAV genomes carrying 11 different enhancers active in mouse brain. We developed spatial genomics methods to identify and localize AAV genomes and their concatemeric forms in cultured cells and in tissue, and we demonstrate here that transcriptional crosstalk is dependent upon concatemer formation. Finally, we leveraged transcriptional crosstalk to drive expression of a 3.2-kb Cas9 cargo in a cell-type-specific manner with systemically administered engineered AAVs, and we demonstrate AAV-delivered, minimally invasive, cell-type-specific gene editing in wild-type mice that recapitulates known disease phenotypes.
Copyright and License
© 2025, The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Acknowledgement
We thank the entire Gradinaru laboratory for careful review and helpful discussions, especially C. Oikonomou for thorough review of the manuscript. We thank P. Anguiano for excellent administrative assistance. We thank A. Askary at the University of California, Los Angeles, for help in implementing the Zombie method for detection of AAV genomes. This work was primarily supported by grants from the National Institutes of Health (Pioneer 5DP1NS111369 and Brain Armamentarium UF1MH128336 to V.G.), the CZI Neurodegeneration Challenge Network (V.G.) and Aligning Science Across Parkinson’s (ASAP-020495 to V.G.) through the Michael J. Fox Foundation for Parkinson’s Research. G.M.C. was supported by a PGS-D from the National Science and Engineering Research Council of Canada. For the purpose of open access, the author has applied a CC BY public copyright license to all Author Accepted Manuscripts arising from the submission.
Data Availability
All sequences of primers, probes, sgRNAs and other sequence elements are provided in Supplementary Table 3. Images of brain tissue that are quantified in this work are deposited in the Brain Image Library (https://doi.org/10.35077/g.1163). Tabular datasets and behavior videos supporting the conclusions of this work are available on Zenodo (https://doi.org/10.5281/zenodo.13952929)89. All other data that support the findings of this study are available from the corresponding authors upon reasonable request.
Conflict of Interest
V.G. is a co-founder and board member of Capsida Biotherapeutics, a fully integrated AAV engineering and gene therapy company. The remaining authors declare no competing interests.
Supplemental Material
- Supplementary Data Figs. 1–3, captions for Supplementary Videos 1 and 2 and Supplementary Table 1
- Representative videos of narrowing beam crossing performance for animals in ubiquitous SaCas9 condition. For display purposes, videos are trimmed to show crossing of a 2.5-cm-wide segment of beam. The entire length of the beam was used for data analysis. Videos show the same animals pre-injection and 4 weeks post-injection.
- Representative videos of narrowing beam crossing performance for animals in crosstalk-mediated PC-specific SaCas9 condition. For display purposes, videos are trimmed to show crossing of a 2.5-cm-wide segment of beam. The entire length of the beam was used for data analysis. Videos show the same animals pre-injection and 4 weeks post-injection.
- Supplementary table listing novel plasmids deposited to Addgene (tab 1) and key resources used in the experiments (tab 2).
- Supplementary table listing key DNA sequences used in the experiments, including AAV titering primers (tab 1), ddPCR primer and probe sets (tab 2), FISH probes (tab 3), Zombie FISH probes (tab 4), sgRNA sequences (tab 5) and other sequence elements, such as promoters and enhancers (tab 6).
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Additional details
- National Institute of Mental Health
- UF1MH128336
- Aligning Science Across Parkinson's
- ASAP-020495
- Chan Zuckerberg Initiative (United States)
- Neurodegeneration Challenge Network -
- Natural Sciences and Engineering Research Council
- National Institutes of Health
- Pioneer 5DP1NS111369
- Accepted
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2025-01-17Accepted
- Available
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2025-03-20Published online
- Caltech groups
- Division of Biology and Biological Engineering (BBE)
- Publication Status
- Published