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

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.