- Gene therapy, the expression in cells of genetic material with therapeutic activity, has emerged as a promising approach for the treatment or prevention of human diseases. At the present time, major somatic gene-transfer approaches employ either viral or nonviral vectors. Nonviral vectors are less efficient at introducing and maintaining foreign gene expression, but have the profound advantage of being nonpathogenic and nonimmunogenic. In this study, we aimed to develop an efficient nonviral gene delivery system in which low-frequency ultrasound (LFUS) was applied to enhance gene expression of polyplexes formed with poly(2-dimethylaminoethyl methacrylate) and plasmid encoding for green fluorescent protein. Ultrasound (US), and in particular LFUS, can cause temporary membrane permeabilization and thereby enhance drug and gene entrance into viable cells. We evaluated possible additional favorable effects of LFUS on the polyplex transfection process, such as overcoming intracellular barriers. We found that pDMAEMA protected the plasmid DNA from ultrasonic degradation. Atomic force microscopy analysis also confirmed that the LFUS did not change the polyplexes’ morphology. We also attained an insight into the structure of polyplexes during LFUS exposure and found that LFUS induced a temporary partial detachment between the polymer chains and the plasmid. In addition, LFUS application on ovarian carcinoma cells transfected with the polyplexes induced a 27% enhancement in transfection efficiency. Based on these results, we propose that LFUS enhances the decomplexation of the polyplexes, and therefore, can be used to optimize transfection efficiency.