The long-term goal of this project is to develop novel systems/methodologies for improving electric field- mediated gene delivery in vivo. This delivery technology, also known as electrotransfection, has been used in gene therapy and DNA vaccination. A challenge for the technology is low efficiency in gene transfer, especially in vivo applications. To improve electrotransfection efficiency (eTE), many studies have been devoted to optimization of electric field parameters (e.g., pulse shape and field strength) for improving transient pore creation in cell membrane (i.e., electroporation) or plasmid DNA (pDNA) transport through the pores via electrophoresis. However, the improvement in eTE has now reached a plateau. One of the key barriers to further improvement is the lack of understanding of pDNA transport pathways in cells. Recent studies have shown that electrotransfection depends on formation of stable complexes between pDNA and plasma membrane induced by applied electric field, and that eTE can be significantly reduced by treating cells with inhibitors of endocytosis. Furthermore, the preliminary data in this proposal showed that knocking down expression of genes involved in endocytosis could reduce eTE in both tumor and normal human primary cells. These observations suggest that completely new strategies need to be developed for further improving eTE. The objective of the proposed study is to determine pathways for intracellular transport of pDNA in electrotransfection. The overall hypothesis is that adsorptive endocytosis is a key pathway for transport of membrane-bound pDNA in electrotransfection. The rationale for the study is that manipulation of new pathways for pDNA transport can lead to development of completely new strategies for improving eTE. The hypothesis will be tested through a systematic investigation of mechanisms that can influence pDNA transport via endocytic pathways in cultured cells (Specific Aim 1). By understanding the mechanisms, the project will develop novel strategies for improving eTE in cultured cells (Specific Aim 2) and three tissues in vivo: normal subcutaneous, normal muscle, and solid tumor (Specific Aim 3). The investigation will use fluorescent markers to label pDNA, cell membrane, and intracellular vesicles for co-localization analysis. The endocytic pathways will be selectively blocked by pharmaceutical inhibitors, dominant-negative mutants, or small interfering RNA (siRNA) that can knock down expression of specific genes in endocytic pathways. The study will quantify dynamic interactions between pDNA and membrane as well as pDNA distributions in the cytoplasm at different time points after electric field application. To facilitate in vivo studies f mechanisms, a unique enabling platform will be developed, which allows non-invasive observation of pDNA and cells in subcutaneous and tumor tissues at high spatial and temporal resolutions. This integrated research is significant and innovative because it will lead to better understanding of pDNA transport mechanisms and development of completely new strategies for improving eTE in vivo, which are critical for clinical applications of electrotransfection.

Public Health Relevance

Gene therapy holds a great promise for treating diseases in patients. One of its current challenges is the lack of safe and efficient methods for gene delivery The goal of the proposed research is to develop novel strategies for improving electric field-mediated gene delivery.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Gene and Drug Delivery Systems Study Section (GDD)
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Okita, Richard T
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Duke University
Biomedical Engineering
Schools of Engineering
United States
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Wang, Liangli; Miller, Sara E; Yuan, Fan (2018) Ultrastructural Analysis of Vesicular Transport in Electrotransfection. Microsc Microanal 24:553-563
Cervia, Lisa D; Chang, Chun-Chi; Wang, Liangli et al. (2018) Enhancing Electrotransfection Efficiency through Improvement in Nuclear Entry of Plasmid DNA. Mol Ther Nucleic Acids 11:263-271
Cervia, Lisa D; Yuan, Fan (2018) Current Progress in Electrotransfection as a Nonviral Method for Gene Delivery. Mol Pharm 15:3617-3624
Mao, Mao; Wang, Liangli; Chang, Chun-Chi et al. (2017) Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection. Mol Ther 25:803-815
Cervia, Lisa D; Chang, Chun-Chi; Wang, Liangli et al. (2017) Distinct effects of endosomal escape and inhibition of endosomal trafficking on gene delivery via electrotransfection. PLoS One 12:e0171699
Chang, Chun-Chi; Mao, Mao; Liu, Yang et al. (2016) Improvement in Electrotransfection of Cells Using Carbon-Based Electrodes. Cell Mol Bioeng 9:538-545
Chang, Chun-Chi; Wu, Mina; Yuan, Fan (2014) Role of specific endocytic pathways in electrotransfection of cells. Mol Ther Methods Clin Dev 1:14058