Electroporation-mediated gene delivery

Abstract

Electroporation has been used extensively to transfer DNA to bacteria, yeast, and mammalian cells in culture for the past 30 years. Over this time, numerous advances have been made, from using fields to facilitate cell fusion, delivery of chemotherapeutic drugs to cells and tissues, and most importantly, gene and drug delivery in living tissues from rodents to man. Electroporation uses electrical fields to transiently destabilize the membrane allowing the entry of normally impermeable macromolecules into the cytoplasm. Surprisingly, at the appropriate field strengths, the application of these fields to tissues results in little, if any, damage or trauma. Indeed, electroporation has even been used successfully in human trials for gene delivery for the treatment of tumors and for vaccine development. Electroporation can lead to between 100 and 1000-fold increases in gene delivery and expression and can also increase both the distribution of cells taking up and expressing the DNA as well as the absolute amount of gene product per cell (likely due to increased delivery of plasmids into each cell). Effective electroporation depends on electric field parameters, electrode design, the tissues and cells being targeted, and the plasmids that are being transferred themselves. Most importantly, there is no single combination of these variables that leads to greatest efficacy in every situation; optimization is required in every new setting. Electroporation-mediated in vivo gene delivery has proven highly effective in vaccine production, transgene expression, enzyme replacement, and control of a variety of cancers. Almost any tissue can be targeted with electroporation, including muscle, skin, heart, liver, lung, and vasculature. This chapter will provide an overview of the theory of electroporation for the delivery of DNA both in individual cells and in tissues and its application for in vivo gene delivery in a number of animal models.

Keywords: Electric field; Electropermeabilization; Electroporation; Endocytosis; Intracellular trafficking; Physical methods; Transfection; Vaccines.

Figure 1. Model for electropermeabilization and electroporation

(A) The presence of an applied electric field induces movement of and redistribution of macromolecular dipoles within and outside the cell, resulting in accumulation of charges across the membrane. Once the transmembrane potential exceeds the dielectric strength of the membrane, transient permeation events occur, generating small hydrophilic pores that stabilize and coalesce into larger pores to allow movement of large molecules such as DNA. (B) The permeabilization of the cell induced by the electric field occurs differentially with respect to pore size: a number of small pores are formed at both poles of the cell, but larger pores capable of allowing DNA entry only form at the anode-facing pole. Modified from Somiari et al. (2000) and Escoffre, Rols, et al. (2011).

Figure 2. Molecular dynamics solution of the formation of hydrophilic water channels in a membrane in response to an applied electric field

Snapshots of the time evolution of water–lipid–water configurations under an external electric field of 500 MV/m. Panels (left to right) are 5.8, 6.7, and 7.3 ns from the start of the simulation with both water molecules (oxygen—red, hydrogen—gray) and lipid molecules (phosphorus—yellow, nitrogen—blue, lipid tail groups—silver) displayed. Reprinted with permission from Tokman et al. (2013). (For interpretation of the references to color in this figure legend see the color plate.)

Figure 3. Postelectroporation trafficking of plasmids to the nucleus during gene transfer

Following electropermeabilization of the membrane, plasmids may enter the cell by either endocytosis and/or direct entry into the cytosol (Rosazza et al., 2013; Rosazza et al., 2011). The initial trafficking events near the cell surface appear to involve actin and actin-based movement. Once through the cortical actin layer and free in the cytoplasm, plasmids are rapidly complexed by a number of DNA-binding proteins present in the cytoplasm which in turn bind to other proteins to form large protein–DNA complexes (Badding et al., 2013). Transcription factors bound to DNA interact with importin β and other proteins that in turn link the complex to dynein for movement along microtubules to the nucleus where it falls apart at the nuclear periphery (Badding et al., 2012). Nuclear entry is then mediated by importin β in a sequence- and importin-dependent manner. (For interpretation of the references to color in this figure see the color plate.)

Figure 4. Examples of electrodes for electroporation

(A) Penetrating, two-needle arrays. (B) Nonpenetrating parallel needles (Genetrode electrodes, Genetronics, San Diego, CA, USA). (C) Plate electrodes (Tweezertrodes, BTX, Hollister, MA). (D) Cartoon of a balloon catheter-based electrode for delivery of DNA and electroporation. (E) Spoon electrode for vascular electroporation. (F) Caliper-mounted plate electrodes. (G) Conformable defibrillator pads for electroporation (arrow). (H) Multielectrode array (R. Heller, personal communication). (For interpretation of the references to color in this figure see the color plate.)