Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 115, Issue 46, Pages E10907-E10914Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1809671115
Keywords
intracellular delivery; T cell engineering; microfluidics; cell therapy
Categories
Funding
- Clinical and Translational Science Awards [U54-TR001012]
- National Science Foundation [1555789]
- National Institutes of Health [R44GM116312]
- Div Of Industrial Innovation & Partnersh
- Directorate For Engineering [1555789] Funding Source: National Science Foundation
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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-( )gamma 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.
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