Engineering novel AAV capsids by global de-targeting and subsequent muscle-specific tropism in mice and NHPs

Engineering novel AAV capsids by global de-targeting and subsequent muscle-specific tropism in mice and NHPs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Engineering novel AAV capsids by global de-targeting and subsequent muscle-specific tropism in mice and NHPs Huapeng Li, Yue Pan, Yujian Zhong, Huan Chen, Youwei Zhang, Zhiyong Dai, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6674722/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Recombinant adeno-associated viral (rAAV) vectors are a potent tool, but their clinical application is restricted by insufficient target tissue transduction and liver toxicity. We employed a novel two-step engineering strategy to create novel rAAV capsids with global tissue de-targeting, then produced strong tissue-specific expression by adding a peptide sequence. We created a novel capsid, AAV.Zero1, with globally de-targeted transduction by loop swapping domains from AAV9 into AAV2. Making an R585A substitution (AAV.Zero2) re-targeted tissues but deleting residues 585–587 (AAV.Zero3) abrogated transduction. Inserting a myogenic peptide into AAV.Zero3 produced a novel capsid (AAV.eM) with strong muscle-specific transgene expression while maintaining minimal off-target expression, including in liver, which was conserved in two mouse strains and non-human primates. AAV.eM showed similar expression as the leading myotropic vector MyoAAV.4A but had a more favorable safety profile. Importantly, AAV.eM was able to functionally rescue a mouse model of Duchenne Muscular Dystrophy following systemic delivery of a micro-dystrophin gene. Thus, AAV.eM is an improved myotropic rAAV capsid that de-targets other tissues, especially the liver, and proof-of-concept for a platform to create capsids with specific properties that translate across species by addition of peptides onto low transduction backbones. Biological sciences/Biotechnology/Gene delivery/Genetic vectors Biological sciences/Biotechnology/Gene therapy rAAV Capsid screening Liver de-targeting Myo-tropism Gene therapy Full Text Additional Declarations Yes there is potential Competing Interest. H.L., Y.P., Y.Z., H.C., Y.Z., Z.D., J.C., K.T., X.C., D.Q., L.S., X.T., Y.F., Y.B., Z.Z., and Z. Y. are employees of PackGene Biotech Inc. PackGene has filed patent applications related to the subject matter of this paper: WO2024138809A1, WO2024138811A1, PCT/CN2025/084668, PCT/CN2025/085186. G.G. is a scientific cofounder of Voyager Therapeutics, Adrenas Therapeutics and Aspa Therapeutics and holds equity in these companies. Supplementary Files SupplementaryTable1.PG016PG017PG018rank.xlsx dataset 1 SupplementaryTable2.Biophysicalstability.xlsx dataset 2 SupplementaryTable3.Primers.xlsx dataset 3 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6674722","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":460897350,"identity":"4f25943b-779f-49e4-9d44-ed0016c75ab0","order_by":0,"name":"Huapeng 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