Composite

Part:BBa_K4040018

Designed by: Tong Yin   Group: iGEM21_NMU_China   (2021-09-21)
Revision as of 12:29, 21 September 2021 by Ytong (Talk | contribs)


Synthetic Receptor CAR-MERTK

Background and structure of the CAR-MERTK

Previous studies have shown that the MER tyrosine kinase (MERTK) expressed on the cytomembrane of the macrophages can not only mediate the phagocytosis of apoptotic cells and virus particles, but at the same time reduce the secretion of cytokines[1]. Therefore, we designed a chimeric receptor, CAR-MERTK, to redirect macrophage MERTK signaling to engulf viruses while circumventing significant cytokines release. Our CAR-MERTK consists of three domains, an αS-scFv extracellular domain targeting the spike (S) protein of the SARS-CoV-2, a CD8 hinge and transmembrane domain as the scaffold, and an intracellular domain derived from MERTK to acquire the magical function of the MERTK. Also, there is a Myc tag gene making the lentiviral transcription efficiency detectable.

Figure1.Vector maps of tested CAR designs and schematics showing the structures of CARs.

The function of CAR-MERTK

The functions of a receptor depend highly on its structure. Since the CAR-MERTK is adapted from both MERTK and CAR, we hypothesize that the receptor can not only recognize the SARS-CoV-2 in a targeted manner but also engulf the SARS-CoV-2 with low inflammatory factors secretion.

Characterization of the CAR-MERTK

1.Validation of the CAR-MERTK expression on the macrophages

We choose the lentiviral technology to transfect CAR-MERTK to human macrophages THP-1 cells. The CAR-MERTK gene was inserted to a third-generation lentiviral vector in which the CMV promotor was replaced by the EF-1α promoter to enhance the expression efficiency. We next used flow cytometry to confirm the expression of the CAR-MERTK on the macrophages by detecting the Myc tag. The results showed that the lentiviral vector transfected THP-1 cells with high efficiency(Figure 2).

Figure2. Membrane-bound CAR expression. Forty-eight hours after retroviral transduction, the expression of synthetic receptors on THP-1 cells was detected by staining with an anti-MYC antibody, followed by flow cytometry analysis. Untransduced THP-1 cells were used as a negative control. The histograms shown in black correspond to the isotype controls, whereas the red histograms indicate positive fluorescence.

2.CAR-MERK macrophages can engulf SARS-CoV-2 S pseudotyped virions

After confirming the expression of CAR-MERTK on the macrophages, we next tested whether the CAR-MERTK macrophages would perform the phagocytosis function as we expected. We first cocultured the CAR-MERTK macrophages with the SARS-CoV-2 S pseudotyped viruses, which could be operated in class-2 biosafety cabinets. Then, the level of S protein in macrophages were measured through flow cytometry to indicate the amount of pseudotyped virions engulfed. As is shown in the diagram(Figure 3), the phagocytosis of the four different CAR-macrophages, including the CAR-MERTK, CAR-MEGF10, CAR-γ , CAR-ζ , but not the truncated CAR (CARΔ), is significantly higher than the UTD group. The results suggested that intracellular domain played an important role in phagocytosis. The low phagocytic score of the CARΔ demonstrated that the phagocytosis was not merely mediated by the affinity of αS -scFV.

Figure3. The uptake of pseudotyped virions by UTD and CAR macrophages was analyzed by flow cytometry. Different cell lines were stained with an anti-S primary Ab. The histograms shown in black correspond to the isotype controls, whereas the red histograms indicate positive fluorescence. Data are reported as the phagocytic score (% positive cells x MFI, right panel).

3.The inflammatory reaction can be blocked when the CAR-MERTK macrophages engulf the SARS-CoV-2 S pseudotyped virions

CAR-MERTK macrophages do not kill the S protein expressing cells, alleviating the inflammatory damage to the body: From the previous study we knew that the S protein would be expressed on the surface of the alveolar epithelial cell after infected by the SARS-CoV-2. There would be extra damage if the CAR-MERTK macrophages still attack the S protein expressing alveolar epithelial cells, aggravating the lung injury. But as mentioned before, the CAR-macrophages are expected to engulf the SARS-CoV-2 in a silent and less harmful way. To demonstrate, we first constructed a targeted cell expressing S protein called Spike-bearing 293 cells through the lentiviral transfection. Then, we cocultured the CAR-MERTK macrophages with the Spike-bearing 293 cells and measured phagocytosis. The results indicated that CAR-MERTK macrophages would not damage the Spike-bearing 293 cells and thus proved the potential safety of our CAR-MERTK macrophages(Figure 4).

4.CAR-MERTK macrophages engulfed the SARS-CoV-2 S pseudotyped virions without increasing inflammatory release

Cytokine release syndrome (CRS) is an important cause of death in severe Covid-19 patients and is a common adverse effect in CAR-T therapies. So, our next goal was to confirm that the CAR-MERTK macrophages can engulf the SARS-CoV-2 viruses in a low inflammatory manner. As the results showed, the cytokines released, including the GM-CSF, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, and IFN-γ, were not significant in the CAR-MERTK macrophages group(Figure 5).

References

[1] Bhattacharyya S, Zagórska A, Lew ED, Shrestha B, Rothlin CV, Naughton J, Diamond MS, Lemke G, Young JA. Enveloped viruses disable innate immune responses in dendritic cells by direct activation of TAM receptors. Cell Host Microbe. 2013 Aug 14;14(2):136-47.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2762
    Illegal XhoI site found at 344
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 952
    Illegal NgoMIV site found at 1288
    Illegal NgoMIV site found at 1825
    Illegal NgoMIV site found at 1891
    Illegal NgoMIV site found at 2050
    Illegal AgeI site found at 440
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1168
    Illegal BsaI.rc site found at 1432
    Illegal BsaI.rc site found at 2113
    Illegal BsaI.rc site found at 2587
    Illegal SapI.rc site found at 1989
    Illegal SapI.rc site found at 2178


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