A Dual-Viral Delivery Platform Enables Efficient Site-Specific Integration of Therapeutic-Length Genes in Human Primary Stem Cells - PubMed
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- #CRISPR Delivery
- #Gene Therapy
- #Stem Cell Engineering
- Developed a dual-viral delivery system (TIVID) combining virus-like Cas9 edit particles (VICEPs) and integrase-deficient lentiviral vectors (IDLVs) for site-specific gene integration.
- Achieved high knock-in efficiency (65% ± 5%) in human iPSCs and 20% efficiency for a 7.1 kb HBB-GFP cassette in erythroid progenitor cells.
- Enabled delivery of ~6 kb full-length HBB cassette into primary human CD34+ hematopoietic stem/progenitor cells with 5-10% integration efficiency and preserved differentiation capacity.
- Outperformed lentivirus-derived nanoparticles and plasmid-based methods (e.g., >50% vs. <10% in K562 cells, ~20% vs. ~1.5% in iPSCs).
- Reduced early cytotoxicity compared to traditional electroporation, favored mono-allelic integration, and showed enhanced compatibility with primary stem cells.
- Decoupled nuclease and donor delivery to overcome payload constraints and toxicity, supporting ex vivo gene therapy for β-thalassemia and multiallelic disorders.