Plasmid Gene Delivery into Rat Laryngeal Muscle Using Bidirectional Electroporation

Abstract Problem: Nonviral gene delivery systems have advantages over viral delivery systems in several aspects including easy propagation on a large scale with high quality, and low immunogenicity. The major drawback of plasmid DNA as a vector is its low efficiency for gene transfer. In this study we examined the delivery of plasmid DNA into laryngeal muscle using electroporation in both unidirectional and bidirectional modalities to enhance gene transfer in laryngeal muscle. Methods: Using a midline thyrotomy approach in adult Sprague-Dawley rats, EGFP-N3 plasmid vector was injected into the left thyroarytenoid muscle. We compared 3 groups as follows: (1) plasmid injection without electroporation, (2) plasmid injection with unidirectional electroporation, and (3) plasmid injection with bidirectional electroporation. The animals were kept alive for 1 week after the injection. Fresh frozen coronal sections of the larynx were used to assess the maximum number of thyroarytenoid muscle fibers expressing EGFP. Results: All groups undergoing electroporation demonstrated increased EGFP expression in the muscle fibers compared to the group without electroporation. Gene expression by total number of cells was increased 2-fold in the bidirectional group when compared to the unidirectional group. Conclusion: Both unidirectional and bidirectional electroporation significantly improved the gene transfer of naked plasmid DNA in laryngeal muscle, with bidirectional electroporation yielding a 2-fold increase over the unidirectional modality. Significance: Electroporative gene transfer using bidirectional modality may improve our ability to deliver trophic factors into laryngeal muscle at lower field strengths, while avoiding the potential adverse effects of viral vectors. Support: None reported.