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Gene therapy holds great potential for treating genetic disorders by correcting or replacing defective genes. While cationic polymers have emerged as promising carriers for nucleic acids, their high cationic charge density often results in substantial cytotoxicity and incomplete cargo dissociation within cells. To overcome these limitations, we designed cationic charge-shifting polymers by post-polymerization ring-opening reactions of poly(vinyl dimethyl azlactone) (PVDMA) with tertiary amines containing nucleophilic groups (amine, hydroxyl, thiol). This approach yielded tertiary amine-modified polymers with either hydrolytically stable amide bonds or degradable ester/thioester linkages. These modifications enabled control over the rate of cationic-to-anionic charge shifting under different environmental conditions, such as temperature, pH, and linkage type, facilitating the efficient release of nucleic acids from polyplexes. Our studies revealed that polymers with thioester linkages could undergo rapid nucleic acid release via thiol-thioester exchange reactions in response to intracellular concentrations of glutathione (GSH). This charge-shifting mechanism provides a potential trigger for intracellular nucleic acid which could thereby enhance delivery efficiency. To explore this further, we synthesized novel thioester-based cationic monomers that could be directly polymerized to produce thioester-functionalized polycations. These new materials demonstrated significantly higher in vitro mRNA transfection efficiencies compared to conventional polycations, such as PDMAEMA, due to their ability to respond to GSH and release their cargo more effectively. Our ongoing work reveals that optimizing molecular weight and monomer composition is critical for balancing polyplex stability and nucleic acid release. Higher molecular weight polymers formed more stable complexes, while lower molecular weight polymers facilitated more rapid hydrolysis. Copolymerization of thioester-functionalized monomers with hydrophobic thioester-based monomers improved hydrolytic stability without compromising GSH-responsiveness. This approach led to significantly higher transfection efficiencies than non-thioester controls. Our findings underscore the potential of thioester-based charge-shifting polymers as a new class of nucleic acid carriers, offering safer and more effective options for gene therapy applications. These materials could pave the way for advancements in therapeutic delivery systems by providing more precise control over release mechanisms in response to intracellular environments.