Cell engineering with microfluidic squeezing preserves functionality of primary immune cells in vivo

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. Her...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2018-11, Vol.115 (46), p.E10907-E10914
Main Authors: DiTommaso, Tia, Cole, Julie M., Cassereau, Luke, Buggé, Joshua A., Hanson, Jacquelyn L. Sikora, Bridgen, Devin T., Stokes, Brittany D., Loughhead, Scott M., Beutel, Bruce A., Gilbert, Jonathan B., Nussbaum, Kathrin, Sorrentino, Antonio, Toggweiler, Janine, Schmidt, Tobias, Gyuelveszi, Gabor, Bernstein, Howard, Sharei, Armon
Format: Article
Language:English
Subjects:
Bioengineering
Biological Sciences
Biomolecules
Cell Engineering - methods
Complications
Compressing
Dendritic Cells - immunology
Electroporation
Electroporation - methods
Engineering
Genes
Genomes
Homing
Humans
Immune system
Immunology
Interleukin 2
Lymphocytes
Lymphocytes T
Microfluidics
Microfluidics - methods
PD-1 protein
PNAS Plus
Proteins
RNA, Messenger - metabolism
T-Lymphocytes - immunology
Transcription
Transcriptome
Tumors
γ-Interferon
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Summary: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
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1809671115