Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS–STING pathway

Nature Biomedical Engineering volume 8, pages 149–164 (2024)Cite this article

Subjects

Abstract

Viral delivery of DNA for the targeted reprogramming of human T cells can lead to random genomic integration, and electroporation is inefficient and can be toxic. Here we show that electroporation-induced toxicity in primary human T cells is mediated by the cytosolic pathway cGAS–STING (cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase–stimulator of interferon genes). We also show that an isotonic buffer, identified by screening electroporation conditions, that reduces cGAS–STING surveillance allowed for the production of chimaeric antigen receptor (CAR) T cells with up to 20-fold higher CAR T cell numbers than standard electroporation and with higher antitumour activity in vivo than lentivirally generated CAR T cells. The osmotic pressure of the electroporation buffer dampened cGAS–DNA interactions, affecting the production of the STING activator 2′3′-cGAMP. The buffer also led to superior efficiencies in the transfection of therapeutically relevant primary T cells and human haematopoietic stem cells. Our findings may facilitate the optimization of electroporation-mediated DNA delivery for the production of genome-engineered T cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Mitigation of the cGAS–STING pathway is crucial for non-viral dsDNA delivery in human T cells.
Fig. 2: STING restoration promotes cell death in Sting−/− T cells upon DNA challenge.
Fig. 3: STING inhibitors fail to rescue DNA-induced T cell death.
Fig. 4: Identification of an electroporation condition that mitigates cGAS–STING activation.
Fig. 5: B1mix-based electroporation promotes target insertion.
Fig. 6: The osmotic pressure of the electroporation buffer regulates cGAS–DNA interactions and 2′3′-cGAMP production.
Fig. 7: B1mix-based electroporation enables the generation of high-quality functional CAR T cells.
Fig. 8: Isotonic-buffer-mediated electroporation improved T cell viability and targeted insertion by mitigating the surveillance of cGAS–STING.

Similar content being viewed by others

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The unprocessed western blots and the source data for the figures and associated statistics are available from figshare at https://doi.org/10.6084/m9.figshare.23700369. All data generated or analysed during the study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

References

  1. Qiu, H.-Y., Ji, R.-J. & Zhang, Y. Current advances of CRISPR-Cas technology in cell therapy. Cell Insight 26, 100067 (2022).

    Article  Google Scholar 

  2. Escors, D. & Breckpot, K. Lentiviral vectors in gene therapy: their current status and future potential. Arch. Immunol. Ther. Exp. 58, 107–119 (2010).

    Article  CAS  Google Scholar 

  3. Milone, M. C. & O’Doherty, U. Clinical use of lentiviral vectors. Leukemia 32, 1529–1541 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Montini, E. et al. Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration. Nat. Biotechnol. 24, 687–696 (2006).

    Article  CAS  PubMed  Google Scholar 

  5. Ranzani, M. et al. Lentiviral vector–based insertional mutagenesis identifies genes associated with liver cancer. Nat. Methods 10, 155–161 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Cesana, D. et al. Uncovering and dissecting the genotoxicity of self-inactivating lentiviral vectors in vivo. Mol. Ther. 22, 774–785 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Merten, O., Geny-Fiamma, C. & Douar, A. Current issues in adeno-associated viral vector production. Gene Ther. 12, S51–S61 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Ayuso, E., Mingozzi, F. & Bosch, F. Production, purification and characterization of adeno-associated vectors. Curr. Gene Ther. 10, 423–436 (2010).

    Article  CAS  PubMed  Google Scholar 

  10. Clément, N. & Grieger, J. C. Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol. Ther. Methods Clin. Dev. 3, 16002 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Deyle, D. R. & Russell, D. W. Adeno-associated virus vector integration. Curr. Opin. Mol. Ther. 11, 442–447 (2009).

    PubMed  PubMed Central  Google Scholar 

  12. Roth, T. L. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405–409 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  13. Schumann, K. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc. Natl Acad. Sci. USA 112, 10437–10442 (2015).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Cornu, T. I., Mussolino, C. & Cathomen, T. Refining strategies to translate genome editing to the clinic. Nat. Med. 23, 415–423 (2017).

    Article  CAS  PubMed  Google Scholar 

  15. Ishikawa, H., Ma, Z. & Barber, G. N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788–792 (2009).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhang, Z.-D. & Zhong, B. Regulation and function of the cGAS-MITA/STING axis in health and disease. Cell Insight 1, 100001 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Decout, A., Katz, J. D., Venkatraman, S. & Ablasser, A. The cGAS–STING pathway as a therapeutic target in inflammatory diseases. Nat. Rev. Immunol. 21, 548–569 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Chen, Q., Sun, L. & Chen, Z. J. Regulation and function of the cGAS–STING pathway of cytosolic DNA sensing. Nat. Immunol. 17, 1142–1149 (2016).

    Article  CAS  PubMed  Google Scholar 

  19. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013).

    Article  ADS  CAS  PubMed  Google Scholar 

  20. Larkin, B. et al. Cutting edge: activation of STING in T cells induces type I IFN responses and cell death. J. Immunol. 199, 397–402 (2017).

    Article  CAS  PubMed  Google Scholar 

  21. Gulen, M. F. et al. Signalling strength determines proapoptotic functions of STING. Nat. Commun. 8, 427 (2017).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  22. Hu, M. M. & Shu, H. B. Innate immune response to cytoplasmic DNA: mechanisms and diseases. Annu. Rev. Immunol. 38, 79–98 (2020).

    Article  CAS  PubMed  Google Scholar 

  23. Cai, X., Chiu, Y.-H. & Chen, Z. J. The cGAS-cGAMP-STING pathway of cytosolic DNA sensing and signaling. Mol. Cell 54, 289–296 (2014).

    Article  CAS  PubMed  Google Scholar 

  24. Härtlova, A. et al. DNA damage primes the type I interferon system via the cytosolic DNA sensor STING to promote anti-microbial innate immunity. Immunity 42, 332–343 (2015).

    Article  PubMed  Google Scholar 

  25. Zhong, B. et al. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation. Immunity 29, 538–550 (2008).

    Article  CAS  PubMed  Google Scholar 

  26. Mukai, K. et al. Activation of STING requires palmitoylation at the Golgi. Nat. Commun. 7, 11932 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kwon, J. & Bakhoum, S. F. The cytosolic DNA-sensing cGAS–STING pathway in cancer. Cancer Discov. 10, 26–39 (2020).

    Article  CAS  PubMed  Google Scholar 

  28. Wang, H. et al. cGAS is essential for the antitumor effect of immune checkpoint blockade. Proc. Natl Acad. Sci. USA 114, 1637–1642 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Haag, S. M. et al. Targeting STING with covalent small-molecule inhibitors. Nature 559, 269–273 (2018).

    Article  ADS  CAS  PubMed  Google Scholar 

  30. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).

    Article  ADS  CAS  PubMed  Google Scholar 

  31. Michalski, S. et al. Structural basis for sequestration and autoinhibition of cGAS by chromatin. Nature 587, 678–682 (2020).

    Article  ADS  CAS  PubMed  Google Scholar 

  32. Gehl, J. Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol. Scand. 177, 437–447 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Du, M. & Chen, Z. J. DNA-induced liquid phase condensation of cGAS activates innate immune signaling. Science 361, 704–709 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  34. Cuevas-Velazquez, C. L. et al. Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells. Nat. Commun. 12, 5438 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zhang, J. et al. Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature 609, 369–374 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yum, S., Li, M., Fang, Y. & Chen, Z. J. TBK1 recruitment to STING activates both IRF3 and NF-κB that mediate immune defense against tumors and viral infections. Proc. Natl Acad. Sci. USA 118, e2100225118 (2021).

  37. Wu, J., Dobbs, N., Yang, K. & Yan, N. Interferon-independent activities of mammalian STING mediate antiviral response and tumor immune evasion. Immunity 53, 115–126.e5 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Yarmush, M. L., Golberg, A., Sersa, G., Kotnik, T. & Miklavcic, D. Electroporation-based technologies for medicine: principles, applications, and challenges. Annu. Rev. Biomed. Eng. 16, 295–320 (2014).

    Article  CAS  PubMed  Google Scholar 

  39. Zhang, C.-P. et al. Efficient non-viral delivery of macromolecules in human primary hematopoietic stem cells and lymphocytes. J. Mol. Cell. Biol. https://doi.org/10.1093/jmcb/mjad018 (2023).

  40. Lei, L. et al. APOBEC3 induces mutations during repair of CRISPR-Cas9-generated DNA breaks. Nat. Struct. Mol. Biol. 25, 45–52 (2018).

    Article  ADS  CAS  PubMed  Google Scholar 

  41. Chicaybam, L. et al. An efficient electroporation protocol for the genetic modification of mammalian cells. Front. Bioeng. Biotechnol. 4, 99 (2016).

    PubMed  Google Scholar 

  42. Shi, Y.-J. et al. DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9. Mol. Cell 82, 4160–4175.e6 (2022).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank H.-B. Shu for sharing the STING-knockout mouse and vectors, and the core facility of the Medical Research Institute at Wuhan University for technical support. This work was supported by the National Key R&D Program of China (2022YFF1002801, 2019YFA0802801 and 2018YFA0801401), the Ministry of Agriculture and Rural Affairs of China, the National Natural Science Foundation of China (31972936 to Y.Z., 31871345 and 32071442 to H.Y.), the Major Scientific and Technological Project of Hubei Province (2022ACA005 to Y.Z and 2022BCA089 to H.Y.), the Medical Science Advancement Program (Basic Medical Sciences) of Wuhan University (TFJC2018005), the Fundamental Research Funds for the Central Universities (2042022dx0003, 2042022kf1190), the Applied Basic Frontier Program of Wuhan City (to H.Y.) and by start-up funding from Wuhan University (to Y.Z. and H.Y.).

Author information

Author notes

  1. These authors contributed equally: Jing An, Chuan-Ping Zhang, Hou-Yuan Qiu, Hong-Xia Zhang.

Authors and Affiliations

  1. Department of Rheumatology and Immunology, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China

    Jing An, Hou-Yuan Qiu, Yu-Ming Zhang, Cai-Xiang Zhang & Ying Zhang

  2. State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, School of Life Sciences, Yunnan University, Kunming, China

    Chuan-Ping Zhang

  3. Department of Urology, Department of Pathology, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China

    Hong-Xia Zhang, Qiu-Bing Chen, Xin-Lin Lei & Hao Yin

  4. TaiKang Centre for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan, China

    Hao Yin

  5. State Key Laboratory of Virology, Wuhan University, Wuhan, China

    Hao Yin & Ying Zhang

  6. RNA Institute, Wuhan University, Wuhan, China

    Hao Yin

Authors
  1. Jing An
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chuan-Ping Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hou-Yuan Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hong-Xia Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Qiu-Bing Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yu-Ming Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xin-Lin Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Cai-Xiang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hao Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ying Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.Z. conceived, designed and managed the project. J.A. and C.-P.Z. performed most experiments and analysed the data. H.-Y.Q., H.-X.Z., Y.-M.Z., X.-L.L. and Q.-B.C. helped with mouse T cell experiments. C.-X.Z. drew the heat map. H.Y. provided conceptual advice and edited the manuscript. Y.Z. wrote the paper, with inputs from all authors.

Corresponding authors

Correspondence to Hao Yin or Ying Zhang.

Ethics declarations

Competing interests

Y.Z., H.Y., C.-P.Z. and J.A. have filed a patent application on the buffer composition through Wuhan University (PCT International Application No. PCT/CN2021/107047). The other authors declare no competing interests.

Peer review

Peer review information

Nature Biomedical Engineering thanks Jinming Gao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 The cGAS-STING pathway is crucial for DNA-induced cell death in mouse T cells.

a, Fold change of cell number and cell viability at 24 h and 120 h post-EP. Wildtype, Sting–/– or Cgas–/– mouse T cells were electroporated with 1 μg plasmid (3.5 kb). b, Fold change of cell number and cell viability at 24 h and 120 h post-EP. WT or Sting−/− mouse T cells infected with empty vector or indicated STING protein upon DNA electroporation or MOCK electroporation. EP condition for this figure: P3-DN100. Data represent mean ± SD, each dot represents biological replicate, n = 3. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test in a and one-way ANOVA with Dunnett’s multiple comparisons test in b.

Source data

Extended Data Fig. 2 STING inhibitors have little effect on DNA-induced cell death.

a, Fold change of cell number and cell viability at 24 h post-EP in human T cells pretreated with H151 at indicated doses. b, Human monocyte cell line THP-1 were pretreated with H151 at indicated dose prior to HSV-1 stimulation. qPCR analysis of ISG56 and IFNB1 showed dose response towards H151, the human STING inhibitor. c, Fold change of cell number and cell viability at 24 h and 120 h post-EP in mouse T cells pretreated with STING inhibitor C176 or C178. For human T cells, P3-EO115 was used. For mouse T cell, P3-DN100 was used. Data represent mean ± SD, n = 3 from independent experiments in a, n = 1 for b. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test in a.

Source data

Extended Data Fig. 3 The osmotic pressure of the electroporation buffer regulates cellular cGAS-DNA interactions.

a, List of buffer composition and their respective osmolality. b, GFP transfection efficiency in mouse T cells electroporated with indicated buffers listed in a. c-d, Conductivity of electroporation buffers (c) and correlation analysis of the conductivity and transfection efficiency (d). e, (left) Confocal images of cGAS and Cy5-labeled dsDNA co-localization in mouse T cells. (right) Quantification of co-localized foci. f-g, Quantification of the cell diameter (f) and the cytoplasmic volume (g) of mouse T cells incubated under the indicated electroporation buffers for 10 minutes. h-i, Comparison of cell viability (h) and GFP positive cell number (i) using buffers with indicated osmolality between Sting–/–, cGas–/– and wildtype mouse T cells. DN100 program was used for electroporation in mouse T cell. Data represent mean ± SD, n = 3 from independent experiments in b-c, g-i. For e-f, each dot represents one cell and n = 58 (e) and n = 106 (f) were measured from three independent experiments. Statistical significance was determined by one-way ANOVA in e, f and g. and e; two-way ANOVA in h and i.

Source data

Extended Data Fig. 4 Sucrose-based hypertonic buffer led to dose-dependent activation of the cGAS-STING pathway and to enhanced cGAS-DNA interactions.

a, Osmotic pressure of B1mix buffer supplemented with sucrose at increased concentrations. b, Transfection efficiency of mouse T cells in sucrose-based hypertonic buffers. c, qPCR analysis of mouse T cells electroporated with sucrose-based hypertonic buffers. d, MST analysis of the in vitro binding capability of cGAS with Cy5 labeled dsDNA in isotonic buffer: B1mix (305 mOsmol/kg), or sucrose-based hypertonic buffers: B1mix + 0.04 M sucrose (352 mOsmol/kg) and B1mix + 0.17 M sucrose (508 mOsmol/kg). e, In vitro production of 2’3’-cGAMP in the indicated buffers. EP program for the figure: DN100. Data represent mean ± SD, n = 3 in a and c; n = 4 in b, d, e from independent experiments. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test.

Source data

Extended Data Fig. 5 Comparison of in vivo antitumor function of CAR T cells generated with viral or non-viral methods.

a, NCG mice were inoculated with 2 × 105 CD19+ Nalm6 cells 4 days prior to 5 × 105 CAR-T cells injection. Tumor burden measured as bioluminescent signal were quantified at indicated days (n = 5 per group). b, Quantification of tumor burden in a, the average photon count of ventral and dorsal acquisitions per animal at indicated time points were recorded. Each line represents one mouse.

Source data

Supplementary information

Supplementary Information

Supplementary figures and tables.

Reporting Summary

Supplementary Data 1

Source data for the supplementary figures.

Supplementary Data 2

Unprocessed western blots for the supplementary figures.

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

An, J., Zhang, CP., Qiu, HY. et al. Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS–STING pathway. Nat. Biomed. Eng 8, 149–164 (2024). https://doi.org/10.1038/s41551-023-01073-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-023-01073-7

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research