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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 for delivering Cas9\/sgRNA ribonucleoprotein complexes and integrase-deficient lentiviral vectors for delivering HDR donor templates. The TIVID system achieves a knock-in efficiency of 65% \u00b1 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\n                    <jats:italic toggle=\"yes\">AAVS1<\/jats:italic>\n                    locus with 20% efficiency and stable expression. Crucially, we demonstrate that TIVID overcomes stringent packaging constraints to deliver an approximately 6 kb full-length\n                    <jats:italic toggle=\"yes\">HBB<\/jats:italic>\n                    therapeutic cassette into primary human CD34\n                    <jats:sup>+<\/jats:sup>\n                    hematopoietic stem and progenitor cells. This platform achieved 5\u201310% targeted integration efficiency and preserved robust lineage-specific differentiation capacity, demonstrating its potential for treating \u03b2-thalassemia and other multiallelic disorders. In head-to-head comparisons, TIVID outperformed lentivirus-derived nanoparticles (\u223c50% vs. &lt;10% at\n                    <jats:italic toggle=\"yes\">AAVS1<\/jats:italic>\n                    in K562 with M3814) and plasmid-based eePASSIGE in iPSCs (\u223c20% vs. \u223c1.5%). Compared with traditional electroporation delivery, TIVID offers lower early cytotoxicity, promotes predominantly mono-allelic integration, and exhibits enhanced compatibility with primary stem cells. By decoupling nuclease and donor delivery, TIVID circumvents the payload constraints of single-vector systems and the toxicity of physical transfection, providing a robust\n                    <jats:italic toggle=\"yes\">ex vivo<\/jats:italic>\n                    engineering platform for complex gene replacement therapies.\n                  <\/jats:p>","DOI":"10.1177\/10430342261453040","type":"journal-article","created":{"date-parts":[[2026,5,28]],"date-time":"2026-05-28T03:53:01Z","timestamp":1779940381000},"page":"858-875","update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":0,"title":["A Dual-Viral Delivery Platform Enables Efficient Site-Specific Integration of Therapeutic-Length Genes in Human Primary Stem Cells"],"prefix":"10.1177","volume":"37","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-7342-8209","authenticated-orcid":false,"given":"Zhu-Ying","family":"Gao","sequence":"first","affiliation":[{"name":"State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology &amp; Blood Diseases Hospital, Chinese Academy of Medical Sciences &amp; Peking Union Medical College, Tianjin, China"},{"name":"Tianjin Institutes of Health Science, Tianjin, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tian-Lai","family":"Shen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology &amp; Blood Diseases Hospital, Chinese Academy of Medical Sciences &amp; Peking Union Medical College, Tianjin, China"},{"name":"Tianjin Institutes of Health Science, Tianjin, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chong-Yan","family":"Cheng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology &amp; Blood Diseases Hospital, Chinese Academy of Medical Sciences &amp; Peking Union Medical College, Tianjin, China"},{"name":"Tianjin Institutes of Health Science, Tianjin, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi-Dan","family":"Sun","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology &amp; Blood Diseases Hospital, Chinese Academy of Medical Sciences &amp; Peking Union Medical College, Tianjin, China"},{"name":"Tianjin Institutes of Health Science, Tianjin, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li-Ming","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology &amp; Blood Diseases Hospital, Chinese Academy of Medical Sciences &amp; Peking Union Medical College, Tianjin, China"},{"name":"Tianjin Institutes of Health Science, Tianjin, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian-Ping","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology &amp; Blood Diseases Hospital, Chinese Academy of Medical Sciences &amp; Peking Union Medical College, Tianjin, China"},{"name":"Tianjin Institutes of Health Science, Tianjin, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiao-Bing","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology &amp; Blood Diseases Hospital, Chinese Academy of Medical Sciences &amp; Peking Union Medical College, Tianjin, China"},{"name":"Tianjin Institutes of Health Science, Tianjin, 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