Flash nanocomplexation as a scalable production method for therapeutic pdna/lpei nanoparticles

Statement of Purpose: Plasmid DNA (pDNA) nanoparticles synthesized by linear polyethylenimine (lPEI) have shown to be effective gene delivery vehicles for therapeutic applications in animal models. However, a critical barrier to clinical translation lies with producing nanoparticles with optimized physicochemical properties in a scalable and reproducible manner. The method should be validated by reliable characterization standards, with a good level of quality control. We have developed a scalable and controllable manufacturing method for production of shelf-stable pDNA/lPEI nanoparticles. Specifically, we have turned a batch mode (pipet mixing) method into a continuous process using the flash nanocomplexation (FNC) method [1]. We have demonstrated excellent reproducibility for this FNC process under quality assurance and quality control standards at different production batch sizes. The sizes of the FNC pDNA/lPEI nanoparticles were tunable with a PDI lower than 0.2. In addition, the FNC nanoparticles could be reliably lyophilized. The lyophilized formulation preserved the composition, physicochemical properties, and bioactivity of the nanoparticles; and, to date, a stability for at least 9 months in-20°C has been achieved. Furthermore, systemically delivered FNC nanoparticles were well tolerated in mice and a large animal model, and biodistribution, determined by bioluminescence and QPCR respectively, showed the efficacy to deliver into many organs/tissues. In vitro and in vivo toxicity assessment of pDNA/lPEI nanoparticles were studied and demonstrated the safety in the translation. These results confirmed that FNC production could overcome many manufacturing limitations of pDNA/lPEI nanoparticles and open up new opportunities for clinical translation of DNA nanomedicine products.