Is Gene Delivery via Electroporation Leading to Changes in Cell State?

Electroporation — the use of high-voltage electric pulses to introduce DNA/RNA/proteins into cells — has made cellular engineering and gene expression easier than ever imagined. One version of this method, called Localized Electroporation (LEP), pushes boundaries of what is possible by interfacing high aspect ratio nanochannels with cell membranes. Such architecture is a major departure from Bulk Electroporation (BEP), one of the most popular gene manipulation methods used in biology labs. As such LEP presents new opportunities in cell engineering and development of cell-based therapeutics. 

According to recent research from Northwestern Engineering’s Horacio Espinosa, LEP is also less disruptive to sensitive cells. Espinosa is the James N. and Nancy J. Farley Professor in Manufacturing and Entrepreneurship at the McCormick School of Engineering.

Using a platform called the Localized Electroporation Device (LEPD) previously developed by Espinosa’s team on murine neural stem cell gene expression, he and his counterparts found that in comparison with BEP, LEP does not lead to extensive cell death or activation of cell stress response pathways that may affect a cell’s long-term physiology. The team found that BEP induces cell stress response gene expression and activation of pathways related to interferon signaling, pH imbalance, oxidative stress, and apoptosis. Crucially, BEP leads to extensive cell death – less than 70 percent of cells are viable.

While BEP can kill cells, LEP preserves high cell viability at a rate above 90 percent and induces significantly less cell stress response. The reduced cell stress caused by LEP enables multi-day delivery into stem cells with better preservation of cell viability and integrity as compared to BEP.

Because of these traits, LEP could potentially allow electroporation to further scale biotechnology breakthroughs into stem cell production and CRISPR-based cell engineering for clinical applications such as disease diagnostics and development of personalized medicine.

The research, “Single Cell Transcriptomics Reveals Reduced Stress Response in Stem Cells Manipulated Using Localized Electric Fields” was published earlier this month in Materials Today Bio. Along with Espinosa, co-authors include Northwestern’s Prithvijit Mukherjee, Chian-Yu Peng, Tammy McGuire, Jin Wook Hwang, Connor H. Puritz, Nibir Pathak, Cesar A. Patino, Rosemary Braun, and John A. Kessler.

Though more investigation is needed – the LEP process appears much less prone than BEP to perturb cell physiology to an extent that it may lead to unintended cell states and responses – the single cell analysis reported in this paper indicates that LEP significantly reduces the cell stress response as compared to BEP. It also demonstrates its use in multi-timepoint delivery experiments without leading to cell death or physiological changes.

“Our study shows that there is utility of LEP in applications such as cell reprogramming and engineering of therapeutic cells where multi-timepoint delivery can improve the efficacy of the outcome,” Espinosa said. “It may also be useful for studying dynamic cellular processes such as disease progression or differentiation by multi-timepoint non-destructive sampling. Such studies where cells need to be perturbed multiple times are difficult to execute with existing technologies as they lead to significant cell stress response and [cell death].”

Following this investigation, Espinosa and his team are planning to use the LEPD platform for applications that involve cell manipulation via gene editing or multi-timepoint delivery of complexes as required for cell therapies. That includes looking into delivering specific proteins/RNA complexes at different times to control cell function or investigate the developmental trajectory of cells, directing them to become a specific type of cell such as a muscle cell or a nerve cell. The team also anticipates the use of localized electroporation principles in new cell constructs, such instrumented tissue, to nondestructively sample cells at various timepoints to study and better understand how their molecular makeup changes at different time frames.

Espinosa added that “application to synthetic biology studies in mammalian cells is also of interest.”