Site-specific Integration of Therapeutic-length Genes using TIVID System

Site-specific Integration of Therapeutic-length Genes using TIVID System | 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 Site-specific Integration of Therapeutic-length Genes using TIVID System Xiao-Bing Zhang, Zhu-Ying Gao, Tian-Lai Shen, Chong-Ya Cheng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7154020/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 Site-specific integration of large genes in human primary stem cells remains a significant challenge in gene therapy, particularly for treating multiallelic diseases. Gene editing efficiency in primary stem cells is heavily influenced by the delivery strategy, which often faces issues with programmability, efficiency, and specificity. Here, we developed a dual-viral delivery system, Targeted Integration via Virus-like Particles and Integrase-Deficient Lentivirus (TIVID). This system combines virus-like Cas9 Edit Particles (VICEP) for delivering Cas9/sgRNA ribonucleoprotein complexes and Integrase-Deficient Lentiviral Vectors (IDLV) for delivering HDR donor templates. The TIVID system achieves a knock-in efficiency of 70 ± 5% in human induced pluripotent stem cells (iPSCs). In erythroid progenitor HUDEP2 cells, TIVID mediates precise integration of a 7.1 kb HBB-GFP cassette (from cut site to cut site) at the AAVS1 locus with 20% efficiency and stable expression, demonstrating its potential for treating β-thalassemia and other multiallelic disorders. In head-to-head comparisons, TIVID outperformed lentivirus-derived nanoparticles (LVNPs) (~ 50% vs < 10% at AAVS1 in K562 with M3814) and plasmid-based eePASSIGE in iPSCs (~ 20% vs ~ 1.5%). Compared to traditional electroporation delivery, TIVID offers lower cytotoxicity and enhanced compatibility with primary stem cells. In conclusion, TIVID is a precise and efficient platform for large-fragment integration, with broad applications in gene therapy. Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells Health sciences/Medical research/Stem-cell research Physical sciences/Engineering/Biomedical engineering Health sciences/Health care/Therapeutics/Gene therapy/Targeted gene repair Health sciences/Health care/Therapeutics/Stem-cell therapies Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTablesforTIVIDSystem.pdf Table S1-S2 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-7154020","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":501028670,"identity":"fca8ab2e-1da7-499f-b4f8-6105fdb44fa1","order_by":0,"name":"Xiao-Bing 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Gene editing efficiency in primary stem cells is heavily influenced by the delivery strategy, which often faces issues with programmability, efficiency, and specificity. Here, we developed a dual-viral delivery system, Targeted Integration via Virus-like Particles and Integrase-Deficient Lentivirus (TIVID). This system combines virus-like Cas9 Edit Particles (VICEP) for delivering Cas9/sgRNA ribonucleoprotein complexes and Integrase-Deficient Lentiviral Vectors (IDLV) for delivering HDR donor templates. The TIVID system achieves a knock-in efficiency of 70\u0026thinsp;\u0026plusmn;\u0026thinsp;5% in human induced pluripotent stem cells (iPSCs). In erythroid progenitor HUDEP2 cells, TIVID mediates precise integration of a 7.1 kb HBB-GFP cassette (from cut site to cut site) at the AAVS1 locus with 20% efficiency and stable expression, demonstrating its potential for treating β-thalassemia and other multiallelic disorders. In head-to-head comparisons, TIVID outperformed lentivirus-derived nanoparticles (LVNPs) (~\u0026thinsp;50% vs\u0026thinsp;\u0026lt;\u0026thinsp;10% at AAVS1 in K562 with M3814) and plasmid-based eePASSIGE in iPSCs (~\u0026thinsp;20% vs\u0026thinsp;~\u0026thinsp;1.5%). Compared to traditional electroporation delivery, TIVID offers lower cytotoxicity and enhanced compatibility with primary stem cells. 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