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Published September 2023 | Published
Journal Article Open

Spatial transcriptomics for profiling the tropism of viral vectors in tissues


A barrier to advancing engineered adeno-associated viral vectors (AAVs) for precision access to cell subtypes is a lack of high-throughput, high-resolution assays to characterize in vivo transduction profiles. In this study, we developed an ultrasensitive, sequential fluorescence in situ hybridization (USeqFISH) method for spatial transcriptomic profiling of endogenous and viral RNA with a short barcode in intact tissue volumes by integrating hydrogel-based tissue clearing, enhanced signal amplification and multiplexing using sequential labeling. Using USeqFISH, we investigated the transduction and cell subtype tropisms across mouse brain regions of six systemic AAVs, including AAV-PHP.AX, a new variant that transduces robustly and efficiently across neurons and astrocytes. Here we reveal distinct cell subtype biases of each AAV variant, including a bias of AAV-PHP.N toward excitatory neurons. USeqFISH also enables profiling of pooled regulatory cargos, as we show for a 13-variant pool of microRNA target sites in AAV genomes. Lastly, we demonstrate potential applications of USeqFISH for in situ AAV profiling and multimodal single-cell analysis in non-human primates.

Additional Information

© 2023 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/. We would like to thank the Gradinaru laboratory for careful review and helpful discussions, especially Y. Lei for help with cloning, X. Ding for sharing the AAV-VP3 plasmid and C. Oikonomou for review of the manuscript writing. We thank C. T. Miller and his laboratory, especially M. Metke and V. P. Singh, as well as the veterinary staff, C. Tang-wing, V. Ngo and C. Chamberlain, at the University of California, San Diego, for their help on the marmoset study. We thank A. S. Fox and his laboratory as well as the research and veterinary staff at the California National Primate Research Center at the University of California, Davis, for their help in conducting rhesus macaque studies. We thank Y. Zhang for assistance with confocal imaging. We also appreciate the help of L. Cai and his laboratory members (N. Koulena, S. Schindler and C. Cronin) in imaging preliminary data and consulting on instrumentation. This study was supported by grants from the National Institutes of Health (NIH; Pioneer 5DP1NS111369 and BRAIN Armamentarium U01 1UF1MH128336 to V.G.). Additional support includes a NARSAD Young Investigator Grant (28097) from the Brain & Behavior Research Foundation and a Caltech BBE divisional postdoctoral fellowship (M.J.J.); the Natural Sciences and Engineering Research Council of Canada (G.M.C.); a Caltech BBE diversity fellowship (C.R.J.); and Eric and Wendy Schmidt by recommendation of the Schmidt Futures program (J.L.V.) Contributions M.J.J., G.M.C. and V.G. designed the study. M.J.J. developed USeqFISH, conducted experiments, established imaging instrumentation, implemented computational pipelines for image processing and analysis and analyzed the data associated with USeqFISH characterization and its applications to cell cultures/tissue. M.J.J. and G.M.C. designed viral constructs, and G.M.C. performed cloning, viral production and mouse injection. C.R.J. performed experiments for Hexb probe validation and assisted with USeqFISH profiling data collection. X.C. performed AAV-PHP.AX engineering with preliminary characterization and provided the marmoset tissue. M.R.C. provided the rhesus macaque tissue and helped with immunochemistry on the NHP tissue. J.L.V. performed neuronal morphology tracing and helped implement data analysis pipelines. A.Z.W. performed cross-validation analysis of cell type clustering with scRNA-seq data. M.J.J. wrote the manuscript, with input from all authors, and V.G. supervised all aspects of this study. Data availability. All sequences of probes and primers used in this study are provided in Supplementary Tables 2 and 3. We used GenBank genome assemblies (mm10 for mouse, cj1700 for marmoset and mmul10 for rhesus macaque) to build Bowtie2 databases for probe design. We used previously published data65 (Gene Expression Omnibus, GSE71585) in Supplementary Fig. 5b. The vector plasmid used to produce AAV-PHP.AX is available at Addgene (195218). Raw image datasets for pooled screening experiments are deposited in the Brain Image Library (https://doi.org/10.35077/g.529). Other data that support the findings of this study are available from the corresponding author upon reasonable request. Code availability. All custom Python code used in this study and an example dataset to test are available at https://github.com/GradinaruLab/useqfish_probedesign (ref. 87; probe/barcode design for USeqFISH and HCR v3), https://github.com/GradinaruLab/useqfish_imaging (ref. 88; automated imaging and fluidics system control) and https://github.com/GradinaruLab/useqfish_analysis (ref. 89; image processing and data analysis). Competing interests. 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.

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In Press - s41587-022-01648-w.pdf


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Additional details

November 20, 2023
January 9, 2024