Enhancement of Electrotransfection Efficiency Through Understanding of Underlying Biological Mechanisms

Abstract

Gene delivery has great potential to cure diseases. However, the field is currently limited by the ability to introduce genes into cells safely and efficiently. The two main categories for gene delivery are viral and non-viral methods. Viral methods generally have high efficiency for delivering genes to cells, but are limited by serious health risks and the size of genes that they can deliver. Non-viral vectors can overcome these problems. A non-viral method with great potential is electrotransfection, which is a physical method that involves the application of an electric field to introduce plasmid DNA (pDNA) into cells. The technique of electrotransfection has also been referred to as electroporation, electropermeabilization, electrogene transfer, and gene electroinjection in the literature [1]. Currently, electrotransfection is one of the most widely used physical approaches as it is simple to use, safe, and can result in successful delivery in some difficult-to-transfect cell lines, such as immune cells and stem cells. The goal of my research is to understand the largely unknown mechanisms by which the electric field delivers pDNA through the plasma membrane, cytosol, and nuclear membrane, which are largely unknown at present. With greater understanding on the underlying biological mechanisms of electrotransfection, we have been able to improve its efficiency.Improvements in gene delivery methods have great potential for increasing the use and implementation of treatments such as gene therapy as well as techniques such as genome editing. Electrotransfection is a technique that has been widely used for gene delivery in both basic research and clinical applications. However, the efficiency of delivery remains low and unstable compared to viral methods. This is mainly because mechanisms of electrotransfection are still largely unknown. The main limitation that prevents wide-spread usage of electrotransfection in clinical practice is its relatively low efficiency; therefore, methods to enhance efficiency have potential for great impact. Prior enhancements in electrotransfection efficiency (eTE) were achieved by optimizing electric field parameters, including electric field strength, pulse number and duration, and pulse shape (square wave versus exponential decay). These methods do not consider the underlying mechanisms. The aims of this investigation are: (1) to understand mechanisms of pDNA endocytosis underlying electrotransfection, (2) to improve efficiency by overcoming physical barriers during intracellular trafficking and upon reaching the nucleus, and (3) to develop techniques that enhance efficiency by combining chemical delivery methods with electrotransfection.The mechanistic studies focus on intracellular trafficking and transport through the nuclear envelope, as previous studies in our lab and others have examined the role of various endocytic pathways [2-5]. Specifically in relation to electrotransfection, the roles of intracellular trafficking involving endocytosis and the role of the nuclear envelope have been much less studied. To our knowledge, this work is the first examining the role of induction of endosomal escape in relation to electrotransfection. Endosomal escape is commonly believed to enhance chemical delivery methods, but we found it is detrimental when naked pDNA is being delivered. These mechanistic studies also involve studying the manipulation of microtubules to understand how recovery from depolymerization increases eTE by several fold. These are in fact some of the largest increases in eTE that we have observed. Additionally, some small molecules can be added as a pretreatment or even in the pulsing buffer to enhance eTE in a manner that is simple and efficient for the user. Importantly, these molecules mostly consist of sugars that are already FDA approved and some have been used as vaccine adjuvants. We have begun to study the mechanisms by which these molecules increase eTE, but a greater understanding of the mechanisms and what characteristic of these molecules allows them to enhance delivery will be essential to finding additional molecules that may be similar yet able to increase the efficiency further and/or improve viability. Finally, the in vivo studies will be essential to translating these improvements into clinical trials. Thus, the goal of my research has been two-fold: one is to understand the molecular mechanisms of DNA transport through the plasma membrane, cytosol, and nuclear envelope; and the other is to develop new approaches to improve delivery efficiency based on results from the mechanistic study.