Year-end Sale % OFF 
Abstract
The translational potential of cell-based therapies is often limited by complications related to effectively engineering and manufacturing functional cells. While the use of electroporation is widespread, the impact of electroporation on cell state and function has yet to be fully characterized. Here, we use a genome-wide approach to study optimized electroporation treatment and identify striking disruptions in the expression profiles of key functional transcripts of human T cells. These genetic disruptions result in concomitant perturbation of cytokine secretion including a 648-fold increase in IL-2 secretion (P < 0.01) and a 30-fold increase in IFN-γ secretion (P < 0.05). Ultimately, the effects at the transcript and protein level resulted in functional deficiencies in vivo, with electroporated T cells failing to demonstrate sustained antigen-specific effector responses when subjected to immunological challenge. In contrast, cells subjected to a mechanical membrane disruption-based delivery mechanism, cell squeezing, had minimal aberrant transcriptional responses [0% of filtered genes misregulated, false discovery rate (FDR) q < 0.1] relative to electroporation (17% of genes misregulated, FDR q < 0.1) and showed undiminished effector responses, homing capabilities, and therapeutic potential in vivo. In a direct comparison of functionality, T cells edited for PD-1 via electroporation failed to distinguish from untreated controls in a therapeutic tumor model, while T cells edited with similar efficiency via cell squeezing demonstrated the expected tumor-killing advantage. This work demonstrates that the delivery mechanism used to insert biomolecules affects functionality and warrants further study.
Highlights
In a direct comparison of functionality, T cells edited for PD-1 via electroporation failed to distinguish from untreated controls in a therapeutic tumor model, while T cells edited with similar efficiency via cell squeezing demonstrated the expected tumor-killing advantage
Electroporation is a commonly used tool to deliver exogenous material into cells for therapeutic purposes, but the consequences of electroporation-induced disruptions on global gene expression, cytokine production, lineage markers, and in vivo function have not been fully characterized, in the context of primary cells for cell therapy [16, 17]. This is especially true for large macromolecules typically used for cell therapy, such as CRISPR-Cas9 ribonucleoproteins (RNPs) [Cas9 protein precomplexed with guide RNA] or DNA [18]
The electroporation program we identified in this optimization screen is the same T cell-optimized electroporation program that is recommended by Lonza for high-efficiency delivery to unstimulated human T cells
Summary
Electroporation is a commonly used tool to deliver exogenous material into cells for therapeutic purposes, but the consequences of electroporation-induced disruptions on global gene expression, cytokine production, lineage markers, and in vivo function have not been fully characterized, in the context of primary cells for cell therapy [16, 17]. This is especially true for large macromolecules typically used for cell therapy, such as CRISPR-Cas ribonucleoproteins (RNPs) [Cas protein precomplexed with guide RNA (gRNA)] or DNA [18]. Electroporation as a nonviral alternative to deliver gene-engineering material removes risks associated with viral delivery, but the functional consequences of doing so have not been fully examined
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
