The Ins and Outs of Messenger RNA Electroporation for Physical Gene Delivery in Immune Cell-Based Therapy

Abstract

Messenger RNA (mRNA) electroporation is a powerful tool for transient genetic modification of cells. This non-viral method of genetic engineering has been widely used in immunotherapy. Electroporation allows fine-tuning of transfection protocols for each cell type as well as introduction of multiple protein-coding mRNAs at once. As a pioneering group in mRNA electroporation, in this review, we provide an expert overview of the ins and outs of mRNA electroporation, discussing the different parameters involved in mRNA electroporation as well as the production of research-grade and production and application of clinical-grade mRNA for gene transfer in the context of cell-based immunotherapies.

Are Cell-Based Therapies Safe and Effective in the Treatment of Neurodegenerative Diseases? A Systematic Review with Meta-Analysis

“…In addition, the inflammatory microenvironment which is often observed in MS and SCI is unfavourable for CNS repair and can be adapted by the administration of anti-inflammatory cells such as regulatory T cells, which are currently extensively tested in the clinic [ 128 , 129 , 130 ]. These cells can be easily engineered to overexpress neuroprotective factors such as neurotrophins, thereby improving their neuroregenerative and neuroprotective properties [ 131 , 132 ]. Optimizing and combining different therapeutic strategies will possibly bring a solution for neurodegenerative diseases in the future.…”

Engineering of regulatory T cells by means of mRNA electroporation in a GMP-compliant manner

“…To the best of our knowledge, this is the first time Tregs are demonstrated to be engineered using mRNA electroporation. This is especially interesting, since some cell types have been shown to be refractory to electroporation [30]. The introduction of an antigen-specific TCR has the potential to increase the suppressive capacity of Tregs by focusing their arsenal in the same targeted direction.…”

“…However, a number of limitations have been attributed to the use of viral vectors, including host immune responses against the viral vector, potential insertional mutagenesis, the maximum insert size, variability of infection potencies, the laborious viral vector production and introduction, and the elevated laboratory costs because of the requirement for a higher biosafety level [28]. Compared with viral delivery of genes, mRNA represents a safer alternative without the risk of insertional mutagenesis and with lower immunogenicity [29,30]. Additionally, mRNA has some advantages over DNA, mostly because no translocation to the nucleus is needed, with no risk of integration into the genome, while expression levels are easily adjusted with the amount of supplied mRNA, expression is almost instant, and it does not rely on promoter strength [30,31].…”

“…Compared with viral delivery of genes, mRNA represents a safer alternative without the risk of insertional mutagenesis and with lower immunogenicity [29,30]. Additionally, mRNA has some advantages over DNA, mostly because no translocation to the nucleus is needed, with no risk of integration into the genome, while expression levels are easily adjusted with the amount of supplied mRNA, expression is almost instant, and it does not rely on promoter strength [30,31]. Moreover, introduced mRNA results in transient gene expression, being subjected to the natural decay of mRNA, providing an accurate system to control the synthesis of exogenous proteins [30].…”

“…Additionally, mRNA has some advantages over DNA, mostly because no translocation to the nucleus is needed, with no risk of integration into the genome, while expression levels are easily adjusted with the amount of supplied mRNA, expression is almost instant, and it does not rely on promoter strength [30,31]. Moreover, introduced mRNA results in transient gene expression, being subjected to the natural decay of mRNA, providing an accurate system to control the synthesis of exogenous proteins [30]. We previously demonstrated the superiority of mRNA transfer by means of electroporation, in terms of efficiency of gene delivery, compared with passive pulsing or lipofection [32À34].…”