A high-capacity combination of Pluronic L64-Cupping for intramuscular gene delivery

Intramuscular injection of plasmid DNA (pDNA) is a promising approach for gene therapy, but its efficiency is hindered by both extracellular and intracellular barriers. The extracellular matrix (ECM), including collagens and nucleases, obstructs pDNA penetration, while intracellular challenges include crossing the plasma membrane, escaping endosomes, and reaching the nucleus. Though non-viral carriers like polymers and cationic lipids have been developed, they often fail to address both barriers simultaneously, leading to poor gene transfer in vivo. Physical methods exist but may damage tissues and cause patient discomfort. Here, we introduce a Pluronic L64-Cupping (L/C) gene delivery system that enhances pDNA delivery by sequentially overcoming ECM diffusion, membrane permeabilization, and intracellular transfection. After intramuscular injection of the pDNA-Pluronic L64 mixture, negative pressure is applied to the injection site, significantly boosting reporter gene expression and sustaining it for at least 42 days. Additionally, this system effectively induces HBsAb production in mice, offering a safe, efficient, and cost-effective platform for both laboratory and clinical gene therapy applications.