iGEM 2020 — Team SMMU-China

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# Team:SMMU-China/Notebook

Horizontal Timeline | CodyHouse

1. [16 Jan](#0)
2. [28 Feb](#0)
3. [20 Mar](#0)
4. [20 May](#0)
5. [09 Jul](#0)
6. [30 Aug](#0)
7. [15 Sep](#0)
8. [01 Nov](#0)
9. [10 Dec](#0)
10. [29 Jan](#0)
11. [3 Mar](#0)

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1. ## Horizontal Timeline

   *January 16th, 2014*

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2. ## Event title here

   *February 28th, 2014*

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3. ## Event title here

   *March 20th, 2014*

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4. ## Event title here

   *May 20th, 2014*

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5. ## Event title here

   *July 9th, 2014*

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6. ## Event title here

   *August 30th, 2014*

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7. ## Event title here

   *September 15th, 2014*

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8. ## Event title here

   *November 1st, 2014*

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9. ## Event title here

   *December 10th, 2014*

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10. ## Event title here

    *January 19th, 2015*

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11. ## Event title here

    *March 3rd, 2015*

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# Team:SMMU-China/Engineering

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## Research and imagine:

The pathophysiology of systemic damage in severe cases of SARS-CoV-2 is the activation of the inflammatory cascade, also named systemic inflammatory reaction syndrome. So, our team was just wondering whether there is a method to decrease or even eliminate the inflammatory cascade in severe cases of SARS-CoV-2. After studying the related researches, we finally found TAM family receptors expressed in the macrophages play a crucial role in the anti-inflammatory responses of the innate immune responses, ensuring the elaborate balance between over-inflammatory and under-inflammatory phase. Further researches showed that MEGF10, common γ subunit of Fc receptors (FcRγ) and CD ζ subunit of T cell receptor also revealed similar anti-inflammatory capacity.

The function of MEGF10, FcRγ and CD ζ subunit of T cell receptors:

The common characteristic of these three receptors is the corporate Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) in intracellular domain. The cytosolic ITAMs in the intracellular domain of the three receptors will be phosphorylated by Src family kinases when they are stimulated by its ligands S protein. Actually, SARS-CoV-2 is a strong stimulus of these three receptors. The activated ITAMs will consequently expose the SH2 binding domains for SH2 matching domain. SH2 matching domain lies in kinase ZAP70 and Syk of T cells and macrophages respectively. Protein Syk, a phagocytic signaling effector can then active the downstream signaling transduction cascades and initiate the phagocytosis in macrophages eventually. Also, growing researches have indicate that these three receptors can perform anti-inflammatory capacity when the macrophages engulf its prey.

The structure of the TAM family receptors:

There are actually three members in the TAM family, named Tyro3, Axl, and MER. The MER tyrosine kinase (MERTK) is consisted of three parts, an extracellular part containing two Ig superfamily domains and two fibronectin type Ⅲ domains for the recognition of its ligands, a transmembrane part ensuring the signal transduction, and an intracellular part consisting an immunoreceptor tyrosine based inhibitory motif (ITIM) domain and a conserved protein tyrosine kinase (PTK) domain for the initiating of inflammatory-free reaction.

![](https://static.igem.org/mediawiki/2020/7/78/T--SMMU-China--engi.jpg)

The function of MER tyrosine kinase (MERTK):

The primary function of MER tyrosine kinase (MERTK) is reducing the locally and systemically inflammatory reaction when the body was stimulated by some pro-inflammatory cytokines. And the reaction was mediated through the followed pathways. On the one hand, the macrophages equipped with MER tyrosine kinase (MERTK) can phagocytes cell debris in a high efficiency, thus reducing the inflammatory reaction mediated by the cell debris. This mechanism is particularly important in severe cases of SARS-CoV-2, as the systemic inflammatory reaction syndrome is mainly initiated by the cell debris of infected or apoptosis alveolar epithelial cell. After the matching between the extracellular part of MER tyrosine kinase (MERTK) and its ligand spike protein, a marked molecular expressed in SARS-CoV-2, the phagocytosis signaling pathway was initiated. Then, Rac1, a GTP-binding protein assuring the rearrangement of cytoskeletal, can be activated through TAM-phospholipase C γ2 (PLC γ2)-Vav-1-Rac1 and TAM-Src family kinase-Rac1 axes, ensuring the phagocytosis of the infected or apoptosis alveolar epithelial cell debris.

![](https://static.igem.org/mediawiki/2020/5/5b/T--SMMU-China--engi2.jpg)

On the other hand, MER tyrosine kinase (MERTK) can prevent chronic activation of macrophages by attenuating inflammatory pathways through particular pattern recognition receptors and cytokine receptors, reducing the inflammatory factors that macrophages release and the inflammatory cells that macrophages recruit. When the MER tyrosine kinase (MERTK) receptors bind with its ligands, the autophosphorylation site in the intracellular part is activated due to the conformational change. And the inflammatory factor secretion pathway inhibitors, suppressor of cytokine signaling (SOCS) proteins and twist proteins, is highly expressed. The SOCS proteins and twist proteins will further inhibit signal transduction of type I IFN-mediated inflammation reaction and Toll-like receptor (TLR) mediated inflammation, known as the most two famous inflammation reaction pathways.

![](https://static.igem.org/mediawiki/2020/2/2e/T--SMMU-China--engi3.jpg)

Accounting the inflammatory-free function of MER tyrosine kinase (MERTK), MEGF10, FcRγ and CD ζ, it occurs to us whether we can also reduce the inflammatory reaction in severe cases of SARS-CoV-2 by equipping the patients with this CAR-macrophages. And here is the road to pursue our whimsical idea.

## Design:

Chimeric antigen receptors (CARs) are frequently used in synthetic biology. It redirects cellular activity to specific targets and performs nearly the best recognition property. So, we want to construct a CAR with the best affinity to SARS-CoV-2, and at the same time, performs immunologically silent property for virus clearance. The most convenient approach to achieve the first goal is to equip the CAR with an scFv derived from an antibody recognizing the virus spike protein. As for the second part, just exchanging the intracellular part of five generation CAR with the intracellular domain of MERTK will be appropriate. Here comes our silent-CAR: an αS-scFv extracellular domain targeting to the spike(S) protein of the SARS-CoV-2, a CD8 hinge and a CD8 transmembrane domain presented in αCD19 CAR for T cells permitting signal transduction, and an intracellular domain with the same structure of the intracellular part of MER tyrosine kinase (MERTK) acquiring the magical function of MER tyrosine kinase (MERTK). Also, there is a Myc tag gene making the lentiviral transcription efficiency detectable. The other three potential anti-inflammatory receptors, common γ subunit of Fc receptors (CARγ), MEGF10 (CARMEGF10) and CD3ζ (CARζ), were constructed with the same methods at the same time. We also constructed a truncated CAR receptor (CARΔ) to follow the principal of controlled trial.

![](https://static.igem.org/mediawiki/2020/f/f7/T--SMMU-China--engi4.jpg)

## Build:

We choose the lentiviral technology to transfect our CAR-MERTK to human macrophages THP-1 cells. The cDNA containing the CAR-MERTK gene was inserted to a third-generation lentiviral vector in which a CMV promotor was equipped. We next confirmed the expression of CAR-MERTK on macrophage by detecting the fluorescence intensity of co-transfected Myc tag through cytometry technology. The other four CAR-macrophages, CARγ, CARMEGF10, CARζ and CARΔ, were also constructed with the same methods. But the result was not awfully satisfactory. Our experiment faculty reminded us to consider the efficiency of transcription, we then realized the transcription efficiency of these CARs needed to be improved.

## Improve:

Plenty of researches have shown that the EF-1α promoter performs extremely high transcription efficiency, we consequently constructed CAR-MERTK cDNA, CARγ cDNA, CARMEGF10 cDNA, CARζ cDNA and CARΔ cDNA with an EF-1α promoter. And the results are inspiring, we finally obtained the five different kinds of CAR-macrophages!

![](https://static.igem.org/mediawiki/2020/f/f4/T--SMMU-China--engi5.jpg)

## Test:

We first plan to evaluate the SARS-CoV-2 engulfment of CAR-MERK macrophages. It showed that the phagocytosis efficiency of CAR-MERTK macrophages is relevant to the amount of SARS-CoV-2 S pseudotyped virions, or the quantity of S protein inside CAR-MERTK macrophages linearly. So, by detecting the fluorescence intensity through cytometry technology after intracellular staining for the S protein was performed, we finally acquired the phagocytosis efficiency of CAR-MERTK macrophages. (Fig 2b) As the diagram shows, the phagocytosis of the CAR-macrophages, including the CAR-MEGF10, CAR-MERTK, CARγ, CARζ, but not CARΔ, is significantly higher than the controlled groups. The results indicate the four candidate CAR performs the similar SARS-CoV-2 phagocytosis ability and this phagocytosis was not initiated by just the combination between CAR and its ligands.

![](https://static.igem.org/mediawiki/2020/9/9d/T--SMMU-China--engi6.jpg)

And then, we want to select the CAR-macrophages with nearly no inflammatory reaction when phagocytosis the virus. The inflammatory reaction in severe cases of SARS-CoV-2 can actually be divided into two parts, the local inflammatory reaction with damaging to alveoli epithelial and the systemic inflammatory reaction with releasing of cytokines which mediated the damage of distant organs.

To start with, we want to dig out whether the candidate CAR-macrophages can phagocytes the S protein expressing autologous cells in an inflammatory-free way. From the previous report we knew the S protein would be exhibited on the surface of alveolar epithelial cell after it was infected by SARS-CoV-2. To imitate the infected alveoli epithelial cells, we constructed a targeted cell expressing S protein called Spike-bearing 293 cells through lentiviral technology. The existence of intracellular S protein-expressing 293 cells, or the presence of S protein inside the CAR-MERTK macrophages was the key evidence to determine whether there was a phagocytosis of S protein expressing autologous cells. So, by only detecting the fluorescence intensity of intracellular staining S protein, we finally acquire the results: only the CAR-MERTK macrophages, but not CAR-MEGF10 macrophages, CARγ-macrophages, CARζ-macrophages or CARΔ-macrophages, would not phagocyte the Spike-bearing 293 cells (Fig 1c, d)

![](https://static.igem.org/mediawiki/2020/6/67/T--SMMU-China--engi7.jpg)

Next, we want to evaluate the systemic inflammatory reaction of this candidate CAR-macrophages. After researching for the resources, we found GM-CSF, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, and IFN-γ are some important cytokines in severe cases of SARS-CoV-2. After coculturing CAR-macrophages with SARS-CoV-2 peudovirus, we tested the concentration of cytokines mentioned before in the supernatant. As the results showed, the inflammatory factors, including the GM-CSF, IL-1β, IL-2, IL-4, IL-5, were significantly low in all experimental group. However, the IL-6, IL-8, IL-10, TNF-α and IFN-γ, exhibited various lower levels compared to controlled group, but with the most significant decrease in CAR-MERTK macrophages group. (Fig 2c)

![](https://static.igem.org/mediawiki/2020/7/76/T--SMMU-China--engi8.jpg)

## Learn:

Combining the characterization experiment above, all of the four candidate CAR-macrophages performs similar phagocytosis efficiency against SARS-CoV-2. However, the inflammatory reducing capacity differs from each other, among which the CAR-MERTK macrophages exhibits the best inflammatory suppression effects. CAR-MERTK macrophages can not only reduce local inflammatory reaction through exemption of infected alveoli epithelial cells but can also decrease the cytokines secretion to lower the systemic inflammatory reaction.

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E-Mail: igem-smmu@smmu.edu.cn

Phone: +86 021-81870925

© 2020. SMMU. All rights reserved.

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# Team:SMMU-China/Experiments

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:SMMU-China/Results

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# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)