Efficient delivery of gene editors using intein-engineered virus-like particles

Efficient delivery of gene editors using intein-engineered virus-like particles | 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 Efficient delivery of gene editors using intein-engineered virus-like particles Xiuming Liang, Guannan Zhou, Vicky Hou, Houze Zhou, Samantha Roudi, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8719484/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Virus-like particles (VLPs) represent a promising next-generation drug delivery platform. However, conventional VLPs rely on multiple viral components for effective cargo encapsulation and delivery, raising safety concerns. Here, we present a novel strategy to engineer immature VLPs using a self-cleaving intein system. We employed viral Gag proteins as sorting domains, linking cargo proteins to Gag through inteins, thereby eliminating the need for the conventional protease cleavage typically mediated by the gag-pol protein. During VLP biogenesis, intein-mediated cleavage released cargo proteins into the lumen, enabling efficient intracellular delivery when VLP surfaces are pseudotyped with VSV-G. Optimal candidates for delivering Cre recombinase and gene editing tools (Cas9, Cas12a and base editors) were identified by screening various Gag proteins. Notably, these VLPs achieved robust gene editing in primary cells, including naïve and activated T cells, as well as hematopoietic stem and progenitor cells (HSPCs). A single local intracerebroventricular (ICV) infusion of optimized particles induced up to 60% tdTomato expression in the brain regions of reporter mice, while intravenous injection resulted in significant recombination (up to 70%) of a variety of cell types across organs. Collectively, we developed a simplified, efficient VLP platform for intracellular cargo delivery with broad therapeutic potential for gene editing and treatment of human diseases. Biological sciences/Biotechnology/Biomaterials/Drug delivery Biological sciences/Biological techniques/Gene delivery/Genetic vectors VLPs intein Gag scaffold protein intracellular delivery gene editing Full Text Additional Declarations Yes there is potential Competing Interest. O.W., J.Z.N., A.G., and S.E.-A. serve as consultants and stakeholders in Evox Therapeutics Limited, Oxford, United Kingdom. All data supporting this study are provided in the manuscript and Supplementary Information. The other authors declare no competing interests. Supplementary Files Supplementarymaterials.pdf Supplementary figures SupplementaryTable1.docx Supplementary Table 1 SupplementaryTable2.docx Supplementary Table 2 Cite Share Download PDF Status: Under Review 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-8719484","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":586598072,"identity":"6b5a3090-ee38-4b7f-aa77-554c7c381865","order_by":0,"name":"Xiuming Liang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYDACZiBOAGI+EIOhIoGHeC1sYC1niNECA2wggrEtgbBKg+M8phse7qhlYGNnfvbw67w0GQb+wwfwapFs5jG7kXjmONBhbObGsttyeBgk0vBbxc8M0tJ2DOQXM2nJbRVALTwG+L2A0ML+TVpyDlAL//kPxNhSA9Yr+bEB6DCGHLw6gH5hKwNqOcAD1FImzXAsjYdNIg2/wwzOH95282dbnRw///Ftkj9qku35+Q8/wG8NBBwGxyAziGQjRj0Q1IFJxh9EKh8Fo2AUjIKRBQAXqThTQWi4bgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0202-1211","institution":"Karolinska Institutet","correspondingAuthor":true,"prefix":"","firstName":"Xiuming","middleName":"","lastName":"Liang","suffix":""},{"id":586598073,"identity":"4233c92f-333a-4b29-84ff-b20cae124b40","order_by":1,"name":"Guannan Zhou","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Guannan","middleName":"","lastName":"Zhou","suffix":""},{"id":586598074,"identity":"4057bcdd-4ada-48bd-b049-10ea03ad8b69","order_by":2,"name":"Vicky Hou","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Vicky","middleName":"","lastName":"Hou","suffix":""},{"id":586598075,"identity":"dfc6de73-cdef-485b-9627-61b806401094","order_by":3,"name":"Houze Zhou","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Houze","middleName":"","lastName":"Zhou","suffix":""},{"id":586598076,"identity":"225485e0-15e8-4060-bfbc-4320e6c672f4","order_by":4,"name":"Samantha Roudi","email":"","orcid":"https://orcid.org/0000-0003-3923-3410","institution":"Karolinska Institute","correspondingAuthor":false,"prefix":"","firstName":"Samantha","middleName":"","lastName":"Roudi","suffix":""},{"id":586598077,"identity":"eb9866f3-651f-405b-8ceb-6c922599145c","order_by":5,"name":"Juliette Suermondt","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Juliette","middleName":"","lastName":"Suermondt","suffix":""},{"id":586598078,"identity":"5767fe0f-aed3-407b-8a26-285f2891991f","order_by":6,"name":"Oskar Gustafsson","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Oskar","middleName":"","lastName":"Gustafsson","suffix":""},{"id":586598079,"identity":"919b9fdc-fe83-47e7-a0ec-0a11c7f54eb0","order_by":7,"name":"Zheyu Niu","email":"","orcid":"","institution":"Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zheyu","middleName":"","lastName":"Niu","suffix":""},{"id":586598080,"identity":"131f76d5-8c9f-4e8e-ae28-6752fb0ce960","order_by":8,"name":"Doste R. 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However, conventional VLPs rely on multiple viral components for effective cargo encapsulation and delivery, raising safety concerns. Here, we present a novel strategy to engineer immature VLPs using a self-cleaving intein system. We employed viral Gag proteins as sorting domains, linking cargo proteins to Gag through inteins, thereby eliminating the need for the conventional protease cleavage typically mediated by the gag-pol protein. During VLP biogenesis, intein-mediated cleavage released cargo proteins into the lumen, enabling efficient intracellular delivery when VLP surfaces are pseudotyped with VSV-G. Optimal candidates for delivering Cre recombinase and gene editing tools (Cas9, Cas12a and base editors) were identified by screening various Gag proteins. Notably, these VLPs achieved robust gene editing in primary cells, including naïve and activated T cells, as well as hematopoietic stem and progenitor cells (HSPCs). A single local intracerebroventricular (ICV) infusion of optimized particles induced up to 60% tdTomato expression in the brain regions of reporter mice, while intravenous injection resulted in significant recombination (up to 70%) of a variety of cell types across organs. 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