Abstract
Owing to the immunogenicity of adeno-associated viruses (AAVs), gene therapies using AAVs face considerable obstacles. Here, by leveraging ex vivo T-cell assays, the prediction of epitope binding to major histocompatibility complex class-II alleles, sequence-conservation analysis in AAV phylogeny and site-directed mutagenesis, we show that the replacement of amino acid residues in a promiscuous and most immunodominant T-cell epitope in the AAV9 capsid with AAV5 sequences abrogates the immune responses of peripheral blood mononuclear cells to the chimaeric vector while preserving its functions, potency, cellular specificity, transduction efficacy and biodistribution. This rational approach to the immunosilencing of capsid epitopes promiscuously binding to T cells may be applied to other AAV vectors and epitope regions.
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Data availability
The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding author on reasonable request. Source data for the figures are provided with this paper.
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Acknowledgements
We thank J. Nelson and Promega Corp. for generously providing NanoLuc reagents; A. M. Sajib for developing the qPCR assay used to quantify the AAV viral genome; C. Soto for training and helping with the 3D structure of the AAV capsid; A. Bear and N. Bhattarai for help with the AAV-manufacturing protocol; J. Phue for confirming the sequences of the plasmids used in the study; W. C. Smith and H. Qu for helping with size exclusion chromatography with multi-angle static light scattering (SEC-MALS); and G.-C. Hung, A. Sharma and J. Berger for reviewing and providing helpful comments on the manuscript. S.J.B., S.L.W., S.-L.C., S.S.N. and R.M. disclose support from the Intramural Research Program of the Center for Biologics Evaluation and Research (CBER), US Food and Drug Administration. S.J.B. and S.S.N. disclose an appointment to the Research Participation Program at CBER administered by the Oak Ridge Institute for Science and Education through the US Department of Energy and the US Food and Drug Administration.
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S.J.B., M.S., T.T.H., S.S.N., C.B., S.L.W. and S.-L.C. performed the experiments and analysed the results. S.J.B. and R.M. designed the experiments and wrote the manuscript. R.M. supervised the entire project. All authors reviewed and edited the manuscript.
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S.J.B. and R.M. are inventors on a patent application related to this work, filed by the US Food and Drug Administration. US Patent No. 63/486,299 (2023). All other authors declare no competing interests.
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Nature Biomedical Engineering thanks Aravind Asokan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Bing, S.J., Seirup, M., Hoang, T.T. et al. Rational immunosilencing of a promiscuous T-cell epitope in the capsid of an adeno-associated virus. Nat. Biomed. Eng 8, 193–200 (2024). https://doi.org/10.1038/s41551-023-01129-8
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DOI: https://doi.org/10.1038/s41551-023-01129-8
