Advancing Electrotransfection in Nucleic Acid Delivery from Mechanistic Understanding to Therapeutic Innovation

Abstract

Nucleic acid medicines, such as genes carried by non-viral vectors, hold immense promise for therapeutic and vaccination applications due to its safety, versatility, and ease of manufacturing. However, persistent limitations in non-viral gene delivery—including inefficient intracellular trafficking, poor nuclear entry, and insufficient transcriptional activity—have hindered their clinical translation. To address the delivery barriers, this dissertation focuses on electrotransfection (ET), one of the physical methods for gene delivery, and systematically investigates key mechanistic and translational aspects of ET.First, a robust quantitative method combining nuclear isolation and qPCR was developed to precisely measure nuclear plasmid DNA (pDNA) delivery kinetics post-ET, identifying critical parameters influencing nuclear localization and providing a quantitative framework to optimize ET conditions. Secondly, a novel biopolymer-based nanoenhancer platform was engineered to improve pDNA stability and intracellular trafficking by transient lysosomal modulation, significantly enhancing in vivo gene expression and overcoming limitations associated with rapid clearance of traditional small molecule enhancers. Thirdly, high-content analysis of images from confocal microscopy led to the discovery of nuclear translocation and chromatin association of Galectin-9 following ET, revealing an unexpected role in regulating plasmid transcriptional activity and identifying new molecular targets to improve delivery efficiency. Finally, an innovative, AI-driven formulation strategy was employed to develop nanoparticle-encapsulated histone deacetylase inhibitors (HDACi) as rational, non-inflammatory adjuvants for DNA vaccines, significantly boosting immune responses and protection of animals in viral challenge experiments. Together, these studies advance fundamental mechanistic understanding and deliver practical solutions that significantly improve non-viral gene delivery outcomes. This integrative approach offers valuable insights, versatile platforms, and broadly applicable methodologies for future clinical translation of gene therapies and DNA vaccines.