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.

  • Letter
  • Published:

A fully human transgene switch to regulate therapeutic protein production by cooling sensation

Nature Medicine volume 25, pages 1266–1273 (2019)Cite this article

Subjects

Abstract

The ability to safely control transgene expression with simple synthetic gene switches is critical for effective gene- and cell-based therapies. In the present study, the signaling pathway controlled by human transient receptor potential (TRP) melastatin 8 (hTRPM8), a TRP channel family member1, is harnessed to control transgene expression. Human TRPM8 signaling is stimulated by menthol, an innocuous, natural, cooling compound, or by exposure to a cool environment (15–18 °C). By functionally linking hTRPM8-induced signaling to a synthetic promoter containing elements that bind nuclear factor of activated T cells, a synthetic gene circuit was designed that can be adjusted by exposure to either a cool environment or menthol. It was shown that this gene switch is functional in various cell types and human primary cells, as well as in mice implanted with engineered cells. In response to transdermal delivery of menthol, microencapsulated cell implants harboring this gene circuit, coupled to expression of either of two therapeutic proteins, insulin or a modified, activin type IIB, receptor ligand trap protein (mActRIIBECD-hFc), could alleviate hyperglycemia in alloxan-treated mice (a model of type 1 diabetes) or reverse muscle atrophy in dexamethasone-treated mice (a model of muscle wasting), respectively. This fully human-derived orthogonal transgene switch should be amenable to a wide range of clinical applications.

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: Design of a cooling sensation-regulated transgene switch.
Fig. 2: Characterization of the CoolSens gene switch in cells.
Fig. 3: Using insulin production from CoolINS-transgenic HEK-293 cells to control blood glucose homeostasis in a T1D mice model.
Fig. 4: Therapeutic efficacy of an ActRIIB trap protein produced from CoolActRII-transgenic HEK-293 cells in mice with dexamethasone-induced muscle atrophy.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available on reasonable request to the corresponding author (M.F.).

References

  1. McKemy, D. D., Neuhausser, W. M. & Julius, D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416, 52–58 (2002).

    Article  CAS  Google Scholar 

  2. Ausländer, S., Ausländer, D. & Fussenegger, M. Synthetic biology—the synthesis of biology. Angew. Chem. Int. Ed. 56, 6396–6419 (2017).

    Article  Google Scholar 

  3. Lanitis, E. et al. Redirected antitumor activity of primary human lymphocytes transduced with a fully human anti-mesothelin chimeric receptor. Mol. Ther. 20, 633–643 (2012).

    Article  CAS  Google Scholar 

  4. Le Guiner, C., Stieger, K., Snyder, R. O., Rolling, F. & Moullier, P. Immune responses to gene product of inducible promoters. Curr. Gene Ther. 7, 334–346 (2007).

    Article  Google Scholar 

  5. Maus, M. V. et al. T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol. Res. 1, 26–31 (2013).

    Article  CAS  Google Scholar 

  6. Anguela, X. M. & High, K. A. An edible switch for gene therapy. Nat. Biotechnol. 34, 824 (2016).

    Article  CAS  Google Scholar 

  7. Nilius, B. & Szallasi, A. Transient receptor potential channels as drug targets: from the science of basic research to the art of medicine. Pharm. Rev. 66, 676–814 (2014).

    Article  Google Scholar 

  8. Patel, T., Ishiuji, Y. & Yosipovitch, G. Menthol: a refreshing look at this ancient compound. J. Am. Acad. Dermatol. 57, 873–878 (2007).

    Article  Google Scholar 

  9. Zakharian, E., Cao, C. & Rohacs, T. Gating of transient receptor potential melastatin 8 (TRPM8) channels activated by cold and chemical agonists in planar lipid bilayers. J. Neurosci. 30, 12526–12534 (2010).

    Article  CAS  Google Scholar 

  10. Crabtree, G. R. & Olson, E. N. NFAT signaling: choreographing the social lives of cells. Cell 109, S67–S79 (2002).

    Article  CAS  Google Scholar 

  11. Xie, M. et al. Beta-cell-mimetic designer cells provide closed-loop glycemic control. Science 354, 1296–1301 (2016).

    Article  CAS  Google Scholar 

  12. Story, G. M. et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112, 819–829 (2003).

    Article  CAS  Google Scholar 

  13. Bautista, D. M. et al. The menthol receptor TRPM8 is the principal detector of environmental cold. Nature 448, 204 (2007).

    Article  CAS  Google Scholar 

  14. Caterina, M. J. et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816 (1997).

    Article  CAS  Google Scholar 

  15. Asuthkar, S. et al. The TRPM8 protein is a testosterone receptor: II. Functional evidence for an ionotropic effect of testosterone on TRPM8. J. Biol. Chem. 290, 2670–2688 (2015).

    Article  CAS  Google Scholar 

  16. Liu, Y., Charpin-El Hamri, G., Ye, H. & Fussenegger, M. A synthetic free fatty acid-regulated transgene switch in mammalian cells and mice. Nucleic Acids Res. 46, 9864–9874 (2018).

    Article  CAS  Google Scholar 

  17. Rooney, J. W., Hodge, M. R., McCaffrey, P. G., Rao, A. & Glimcher, L. H. A common factor regulates both Th1- and Th2-specific cytokine gene expression. EMBO J. 13, 625–633 (1994).

    Article  CAS  Google Scholar 

  18. Berridge, M. J., Bootman, M. D. & Roderick, H. L. Calcium signalling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4, 517–529 (2003).

    Article  CAS  Google Scholar 

  19. Crabtree, G. R. & Schreiber, S. L. SnapShot: Ca2+–calcineurin–NFAT signaling. Cell 138, 210–210.e1 (2009).

    Article  CAS  Google Scholar 

  20. Muller, M. R. & Rao, A. NFAT, immunity and cancer: a transcription factor comes of age. Nat. Rev. Immunol. 10, 645–656 (2010).

    Article  Google Scholar 

  21. Orive, G. et al. Cell encapsulation: technical and clinical advances. Trends Pharm. Sci. 36, 537–546 (2015).

    Article  CAS  Google Scholar 

  22. Jacobs-Tulleneers-Thevissen, D. et al. Sustained function of alginate-encapsulated human islet cell implants in the peritoneal cavity of mice leading to a pilot study in a type 1 diabetic patient. Diabetologia 56, 1605–1614 (2013).

    Article  CAS  Google Scholar 

  23. Vaithilingam, V. et al. Characterisation of the xenogeneic immune response to microencapsulated fetal pig islet-like cell clusters transplanted into immunocompetent C57BL/6 mice. PLoS ONE 8, e59120 (2013).

    Article  CAS  Google Scholar 

  24. Vegas, A. J. et al. Long-term glycemic control using polymer-encapsulated human stem cell–derived beta cells in immune-competent mice. Nat. Med. 22, 306 (2016).

    Article  CAS  Google Scholar 

  25. Zakrzewski, J. L., van den Brink, M. R. & Hubbell, J. A. Overcoming immunological barriers in regenerative medicine. Nat. Biotechnol. 32, 786–794 (2014).

    Article  CAS  Google Scholar 

  26. Zekorn, T. D. C. et al. Evidence for an antigen-release induced cellular immune response against alginate-polylysine encapsulated islets. Xenotransplantation 2, 116–119 (1995).

    Article  Google Scholar 

  27. Johnson, J. D. The quest to make fully functional human pancreatic beta cells from embryonic stem cells: climbing a mountain in the clouds. Diabetologia 59, 2047–2057 (2016).

    Article  CAS  Google Scholar 

  28. Lecker, S. H., Goldberg, A. L. & Mitch, W. E. Protein degradation by the ubiquitin–proteasome pathway in normal and disease states. J. Am. Soc. Nephrol. 17, 1807–1819 (2006).

    Article  CAS  Google Scholar 

  29. Williams, G. R., Muss, H. B. & Shachar, S. S. Cachexia in patients with cancer. Lancet Oncol. 17, e220 (2016).

    Article  Google Scholar 

  30. Hq Han, X. Z. Novel hybrid ActRIIB ligand trap proteins for treating muscle wasting diseases. European Patent, EP3286206A1 (2015).

  31. Zhou, X. et al. Reversal of cancer cachexia and muscle wasting by ActRIIB antagonism leads to prolonged survival. Cell 142, 531–543 (2010).

    Article  CAS  Google Scholar 

  32. Lach-Trifilieff, E. et al. An antibody blocking activin type II receptors induces strong skeletal muscle hypertrophy and protects from atrophy. Mol. Cell Biol. 34, 606–618 (2014).

    Article  Google Scholar 

  33. Kaufmann, K. B., Buning, H., Galy, A., Schambach, A. & Grez, M. Gene therapy on the move. EMBO Mol. Med. 5, 1642–1661 (2013).

    Article  CAS  Google Scholar 

  34. Orive, G. et al. History, challenges and perspectives of cell microencapsulation. Trends Biotechnol. 22, 87–92 (2004).

    Article  CAS  Google Scholar 

  35. Fischbach, M. A., Bluestone, J. A. & Lim, W. A. Cell-based therapeutics: the next pillar of medicine. Sci. Transl. Med. 5, 179ps177 (2013).

    Article  Google Scholar 

  36. Kobayashi, T. et al. Immune mechanisms associated with the rejection of encapsulated neonatal porcine islet xenografts. Xenotransplantation 13, 547–559 (2006).

    Article  Google Scholar 

  37. Bai, P. et al. A synthetic biology-based device prevents liver injury in mice. J. Hepatol. 65, 84–94 (2016).

    Article  CAS  Google Scholar 

  38. Schukur, L., Geering, B., Charpin-El Hamri, G. & Fussenegger, M. Implantable synthetic cytokine converter cells with AND-gate logic treat experimental psoriasis. Sci. Transl. Med. 7, 318ra201 (2015).

    Article  Google Scholar 

  39. Wang, H., Xie, M., Charpin-El Hamri, G., Ye, H. & Fussenegger, M. Treatment of chronic pain by designer cells controlled by spearmint aromatherapy. Nat. Biomed. Eng. 2, 114–123 (2018).

    Article  CAS  Google Scholar 

  40. Ye, H., Daoud-El Baba, M., Peng, R. W. & Fussenegger, M. A synthetic optogenetic transcription device enhances blood-glucose homeostasis in mice. Science 332, 1565–1568 (2011).

    Article  CAS  Google Scholar 

  41. Boukamp, P. et al. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106, 761–771 (1988).

    Article  CAS  Google Scholar 

  42. Simonsen, J. L. et al. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells. Nat. Biotechnol. 20, 592–596 (2002).

    Article  CAS  Google Scholar 

  43. Heng, B. C. et al. mRNA transfection-based, feeder-free, induced pluripotent stem cells derived from adipose tissue of a 50-year-old patient. Metab. Eng. 18, 9–24 (2013).

    Article  CAS  Google Scholar 

  44. Wieland, M., Auslander, D. & Fussenegger, M. Engineering of ribozyme-based riboswitches for mammalian cells. Methods 56, 351–357 (2012).

    Article  CAS  Google Scholar 

  45. Berger, J., Hauber, J., Hauber, R., Geiger, R. & Cullen, B. R. Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66, 1–10 (1988).

    Article  CAS  Google Scholar 

  46. Schlatter, S., Rimann, M., Kelm, J. & Fussenegger, M. SAMY, a novel mammalian reporter gene derived from Bacillus stearothermophilus alpha-amylase. Gene 282, 19–31 (2002).

    Article  CAS  Google Scholar 

  47. Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101 (2008).

    Article  CAS  Google Scholar 

  48. Mates, L. et al. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat. Genet. 41, 753–761 (2009).

    Article  CAS  Google Scholar 

  49. Fluri, D. A., Kemmer, C., Daoud-El Baba, M. & Fussenegger, M. A novel system for trigger-controlled drug release from polymer capsules. J. Control. Release 131, 211–219 (2008).

    Article  CAS  Google Scholar 

  50. Auslander, D. et al. A synthetic multifunctional mammalian pH sensor and CO2 transgene-control device. Mol. Cell 55, 397–408 (2014).

    Article  CAS  Google Scholar 

  51. Shao, J. et al. Smartphone-controlled optogenetically engineered cells enable semiautomatic glucose homeostasis in diabetic mice. Sci. Transl. Med. 9, eaal2298 (2017).

    Article  Google Scholar 

  52. Fritschy, W. M. et al. The capsular overgrowth on microencapsulated pancreatic islet grafts in streptozotocin and autoimmune diabetic rats. Transpl. Int. 7, 264–271 (1994).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank F. Viana for providing prTRPM8, F. Kühn for providing phTRPM8 and X. Dong for providing pmTRPM8. We also thank M. Daoud-El Baba, J. Jiang and J. Yin for supporting the in vivo work; V. Jäggin, M. Di Tacchio and Y. Zhang for assistance with flow cytometry; T. Lummen for help with time-lapse microscopy and A. Ponti for support with statistical analysis. This work was supported by a personal fellowship from the Chinese Scholarship Council to P.B., by the National Centre of Competence in Research Molecular Systems Engineering and in part by the European Research Council advanced grant (ElectroGene, no. 785800) (both to M.F.). This work was also financially supported by grants from the National Natural Science Foundation of China (no. 31861143016), the Science and Technology Commission of Shanghai Municipality (no. 18JC1411000) and the Thousand Youth Talents Plan of China to H.Y.

Author information

Authors and Affiliations

  1. Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland

    Peng Bai, Ying Liu, Pratik Saxena, Mingqi Xie & Martin Fussenegger

  2. Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China

    Shuai Xue & Haifeng Ye

  3. Département Génie Biologique, Institut Universitaire de Technologie de Lyon 1, Villeurbanne, France

    Ghislaine Charpin-El Hamri

  4. Faculty of Science, University of Basel, Basel, Switzerland

    Martin Fussenegger

Authors
  1. Peng Bai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ying Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shuai Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ghislaine Charpin-El Hamri
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pratik Saxena
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Haifeng Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mingqi Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Martin Fussenegger
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.B. and M.F conceived the project. P.B., Y.L., M.X., P.S., H.Y. and M.F. designed the experiments. P.B., Y.L., M.X., P.S., H.Y. and M.F. analyzed the results. P.B., Y.L., S.X., P.S. and G.C.-E.H. performed the experimental work. P.B., Y.L., M.X., P.S. and M.F. wrote the manuscript.

Corresponding author

Correspondence to Martin Fussenegger.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information: Michael Basson was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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

Extended data

Extended Data Fig. 1 Functional characterization of CoolSens gene switch in vitro

a, Menthol-inducible NFAT translocation on hTRPM8 activation demonstrated with micrographs of both HEK-293 cells cotransfected with HA-NFAT1(4-460)-GFP (PhCMV-NFAT1(4-460)-GFP-pA) and pPB112 (PhEF1α-hTRPM8-pA), and cultivated for 1 h in the absence or presence of 100 µM menthol. Differences between the images are highlighted by arrows. From left to right: phase contrast, fluorescence and overlaid microscope images. The experiments were repeated independently at least three times with similar results. b, Ciclosporin-mediated inhibition of menthol-induced transgene expression demonstrated with SEAP levels in culture supernatants of pPB112/pPB111-transgenic HEK-293 cells induced by menthol (50 µM) and cultivated for 24 h in the presence or absence of ciclosporin. Data presented are mean ± s.d.; n = 3 biologically independent samples. c, SEAP levels in culture supernatants of pPB112/pPB111-transgenic HEK-293 cells cultivated for 24 h in medium containing menthol esters (i), menthol derivatives (ii) or monoterpenes (iii) with a mint-like flavor and capsaicin. Data presented are mean ± s.d.; n = 3 biologically independent samples. d, SEAP levels in culture supernatants of pPB112/pPB111-transgenic HEK-293 cells cultivated for 24 h in medium containing testosterone or menthol. Data presented are mean ± s.d.; n = 3 biologically independent samples. e, SEAP levels in culture supernatants of HEK-293 cells cotransfected with pMX57 (PNFAT3-SEAP-pA) and a plasmid encoding a constitutive expression unit for TRPM8 of human, rat or murine origin, cultivated for 24 h in the presence of 100 µM menthol. Data presented are mean ± s.d.; n = 3 biologically independent samples. f, SEAP levels in culture supernatants of a mixed population between phTRPM8/pMX57-transgenic HEK-293 cells and pPB108/pMX57-transgenic HEK-293 cells cultivated for 24 h at 37 °C following a 1-h stimulation time at 18 °C or 42 °C. Control cells were kept constantly at 37 °C. Data presented are mean ± s.d.; n = 3 biologically independent samples. g, SEAP levels in culture supernatants of pPB112/pPB111-transgenic HEK-293 cells cultivated for 24 h in menthol-free medium after a pre-stimulation with 50 µM menthol for different time periods and subsequent medium exchange. Data presented are mean ± s.d.; n = 3 biologically independent samples.

Extended Data Fig. 2 Orthogonality of CoolSens in vitro.

a, SEAP levels in culture supernatants of HEK-293 cells transfected with plasmids encoding hGPR40 (PhCMV-hGPR40-FLAG-pA), human phTRPM8 (PhCMV-hTRPM8-pA) and pMX57 (PNFAT3-SEAP-pA), and cultivated for 24 h with the respective agonist of each receptor (oleic acid 10 µM, 50 µM; menthol, 50 µM). Data presented are mean ± s.d.; n = 3 biologically independent samples. b, SEAP levels in culture supernatants of HEK-293 cells transfected with plasmids encoding human GPR40 (PhCMV-hGPR40-FLAG-pA), human phTRPM8 (PhCMV-hTRPM8-pA) and pYL1 (PCRE-SRE-NFAT-SEAP-pA), and cultivated for 24 h with the respective agonist of each receptor (oleic acid 10 µM; menthol, 50 µM). Data presented are mean ± s.d.; n = 3 biologically independent samples. c, Insensitivity of CoolSens to endogenous NFAT agonists demonstrated with SEAP levels in culture supernatants of pPB112/pPB111-transgenic HEK-293 cells cultivated for 48 h in medium supplemented with different NFAT agonists at bioactive concentrations (VEGF165 (40 ng ml–1), insulin (100 ng ml–1), angiotensin II (1 µM), l-norepinephrine (100 µM), acetylcholine (2 mM) and IL-6 (1 ng ml–1). KCl, 20 mM, was used as a positive control. Data presented are mean ± s.d.; n = 3 biologically independent samples. d, Relative expression levels of representative NFAT-target genes (see Supplementary Table 2) in CoolSens cells after menthol-triggered activation determined with RT–qPCR. HEK-293 cells, CoolSens cells stimulated by menthol for 1 week or CoolSens cells stimulated by menthol for 5 weeks were harvested for RNA extraction. Data presented are mean ± s.d.; n = 3 biologically independent samples.

Extended Data Fig. 3 Functionality of the CoolSens gene switch in encapsulated cells.

a, Stable HEK-293-derived CoolSens cells were encapsulated into alginate-poly(l-lysine)-alginate microcapsules and cultivated in six-well plates (~5 × 105 cells per well). SEAP expression in the culture supernatants was profiled at 24 h after the addition of menthol (50 µM). All the data are shown as mean ± s.d.; n = 3 independent experiments. b, Stable HEK-293-derived CoolSens cells were encapsulated into hollow-fiber macrocapsules and cultivated in six-well plates (~1 × 105 cells per well). SEAP expression in the culture supernatants was profiled at 24 h after coculturing with menthol (50 µM). All data are shown as mean ± s.d.; n = 4 biologically independent samples. c, Stable HEK-293-derived CoolSens cells were encapsulated into cellulose microcapsules and cultivated in six-well plates (~5 × 105 cells per well). SEAP expression in the culture supernatants was profiled after 24 h in the presence of menthol (50 µM). All data are shown as mean ± s.d. ; n = 3 independent experiments. P values were determined using a two-tailed, Student’s t-test.

Extended Data Fig. 4 Orthogonality of CoolSens in vivo.

a, Effect of typical menthol-containing foods on transgene expression in mice demonstrated with 24-h SEAP levels in the bloodstream of mice harboring intraperitoneal CoolSens implants (2 × 106 cells per mouse) and receiving oral administrations of freshly prepared peppermint tea (100 µl of 10 ml water containing 1 g dry peppermint leaves), peppermint essential oil (100 µl of 1:10 dilution in olive oil) or menthol (0.3 M in olive oil) (positive control). Data are shown as mean ± s.e.m.; n = 5, each dot representing an individual mouse. Statistics by one-way ANOVA test. b, Effect of cold exposure on transgene expression in mice demonstrated with 24-h SEAP levels in the bloodstream of mice harboring subcutaneous CoolSens implants (2 × 106 cells per mouse) and either (i) kept at room temperature (room temperature, n = 6), in a room with constant temperature control at 15 °C (15 °C environment, n = 6), subjected to ice-pack application on the skin for 1 h (local ice bag, n = 5) or given transdermal application of menthol (0.6 M menthol in olive oil/Vaseline) (positive control, n = 6), or (ii) placed in a cold room (4 °C, n = 6) for 3 h. Data are shown as mean ± s.e.m., each dot representing an individual mouse. P values were determined using a two-tailed, Student’s t-test. For the purpose of comparison, the SEAP levels from wild-type mice kept at room temperature (control, Fig. 3a) were replicated. Statistics for (i) were performed by a one-way ANOVA test. c, The 24-h SEAP levels in the bloodstream of mice harboring intraperitoneal CoolSens implants and exposed to menthol vapor for 8 h. Data are shown as mean ± s.e.m.; n = 6, each dot representing an individual mouse. P values were determined using a two-tailed, Student’s t-test between indicated datasets.

Extended Data Fig. 5 Kinetics of CoolINS-mediated protein secretion in vivo.

SEAP levels in the bloodstream sampled at different time points from mice harboring intraperitoneal CoolSens implants and receiving oral administrations of olive oil (− menthol) or menthol (0.15 M in olive oil). Data are shown as mean ± s.e.m., each dot representing an individual mouse (n = 6 mice per group). P values were determined using a two-tailed, Student’s t-test between indicated datasets.

Extended Data Fig. 6 Long-term functionality of CoolINS cells in vivo.

a,b, T1D C57BL/6 mice (n = 7) were implanted with 2 × 106 CoolINS cells intraperitoneally and given oral administration of menthol solution (0.3 M in olive oil) at the indicated time points for 4 weeks. The placebo group received menthol without CoolINS cell implants (n = 5). Statistics were using a two-tailed, Student’s t-test. a, Fasting blood glucose levels were measured at the indicated time points. b, Intraperitoneal glucose tolerance test was performed on day 28. Healthy mice (n = 5) received no treatment. c, Impact of menthol on fasting glycemia in mice. T1D CD-1 mice (n = 6) were treated with topical menthol ointment (0.6 M menthol in olive oil/Vaseline) for 4 d and fasting blood glucose levels were assayed at indicated time points. Statistics were by one-way ANOVA test. Data are shown as mean ± s.e.m.

Extended Data Fig. 7 Long-term functionality of CoolSens cells in vivo.

a, Reversible transgene induction demonstrated with SEAP levels in the bloodstream of C57BL/6 mice harboring peritoneal CoolSens cell capsules and receiving repeated oral administrations of menthol (0.3 M in olive oil) for 5 weeks. Each dot represents an individual mouse (n = 8 mice, day 2 to day 16; n = 6 mice, day 19 to day 30). Data are shown as mean ± s.e.m.; P values were determined using a two-tailed, Student’s t-test between indicated datasets. b, Macrophage depletion enhances transgene expression. One day before implantation of CoolSens cell capsules, C57BL/6 mice were treated with or without clodronate liposomes (single dose of 200 µl intraperitoneally n = 6 mice). Menthol (0.3 M in olive oil) was orally administered to mice once a week before serum SEAP levels were assayed. For the purpose of comparison, the SEAP levels (weeks 1 and 2) from wild-type mice (n = 8 mice) (a) were replicated in b . Data are shown as mean ± s.e.m., each dot representing an individual mouse. P values were determined using a two-tailed, Student’s t-test between indicated datasets. c, Micrographs of cell capsules explanted from mice treated as described in b using peritoneal lavage. Experiments were repeated independently three times with similar results.

Extended Data Fig. 8 Host immune response to designer cell implants.

a–d, Wild-type C57BL/6 mice were implanted intraperitoneally with empty capsules, capsules containing mouse-derived C2C12 cells or capsules containing human-derived HEK-293 CoolSens cells for 3 weeks. a, Micrographs of cell capsules explanted by peritoneal lavage. Red fluorescence indicates CoolSens cells. b, Retrieval efficacy of different cell capsules calculated by the percentage of retrievable microcapsules at week 3 post-transplantation in respect to the initial number of implanted cell capsules (n = 5 mice). Data are shown as mean ± s.e.m., P values were determined using a two-tailed, Student’s t-test between indicated datasets. c, Total count of cells retrieved by peritoneal lavage at week 3 post-implantation (n = 5 mice), Data are shown as mean ± s.e.m. d, FACS analysis of the retrieved peritoneal cells described in c for different cell-surface markers (n = 4 mice). Data are shown as mean ± s.e.m. e–i, Wild-type C57BL/6 mice were implanted intraperitoneally with capsules containing human-derived HEK-293 CoolSens cells for 5 weeks while receiving oral menthol administration (0.3 M in olive oil) once a week (same experimental group as in Extended Data Fig. 7a). Control groups received either no capsules or capsules containing HEK-293 cells lacking the CoolSens circuit. e, Immunological characterization of spleen cell population in mice (n = 4 mice Data are shown as mean ± s.e.m. f, Total count of white blood cells, blood monocytes and blood lymphocytes in mice. White blood cell count (WBC) (n = 6 mice). Data are shown as mean ± s.e.m. g, Serum TNF-α levels in mice. Statistics by one-way ANOVA test. NS (n = 6 mice). Data are shown as mean ± s.e.m. Statistics by one-way ANOVA test. h, Serum IFN-γ levels in mice. Statistics by one-way ANOVA test (n = 6 mice Data are shown as mean ± s.e.m. i, Serum IL-6 levels in mice (n = 6 mice). Data are shown as mean ± s.e.m. Each dot represents an individual mouse.

Extended Data Fig. 9 Characterization of CoolINS-mediated insulin expression in vitro.

a, Insulin expression kinetics determined with ELISA of cell culture medium of 1 × 106 CoolINS cells cultivated in 2 ml menthol-containing medium. Data presented are mean ± s.d.; n = 3 biologically independent samples. b, Functional assessment of insulin activity. Analogous to the assay described in Fig. 4b, phIR/MKp37/pLeo665-transfected HEK-293 cells were cultivated with conditional medium from HEK-293 cells (control) or CoolINS cells with 50 μM menthol (conditional medium) for 24 h, and then NanoLuc production in the supernatant was quantified. Data presented are mean ± s.d.; n = 3 biologically independent samples.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2

Reporting Summary

Supplementary Video

Supplementary Video 1

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bai, P., Liu, Y., Xue, S. et al. A fully human transgene switch to regulate therapeutic protein production by cooling sensation. Nat Med 25, 1266–1273 (2019). https://doi.org/10.1038/s41591-019-0501-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41591-019-0501-8

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