Difference between revisions of "Part:BBa K4175008"

 
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<partinfo>BBa_K4175008 short</partinfo>
 
<partinfo>BBa_K4175008 short</partinfo>
  
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When the concentration of IL-6 is high, the binding of IL-6 with anti-IL-6 scFv (BBa_K4175000) will cause the Notch core domain (BBa_K4175001) undergo intracellular proteolytic cleavage. Consequently, Gal4-KRAB domain (BBa_K2446037) is released into the cytosol and subsequently, the nucleus. The DNA binding domain of Gal4 will bind to the UAS region, while the KRAB domain will inhibit the promoter next to the UAS region. In this way, the expression of the gene regulated by UAS is inhibited.
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===Sequence and Features===
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<partinfo>BBa_K4175008 SequenceAndFeatures</partinfo>
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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[[File:IL6 scfv-Notch-Gal4KRAB.png|thumb|left|600px|<b>Figure 1.</b> The schematic of IL-6_scfv-Notch-Gal4KRAB under low and high serum IL-6 level.]]
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This device incorporates anti-IL-6 scFv (<partinfo>BBa_K4175000</partinfo>), the Notch core domain (<partinfo>BBa_K4175001</partinfo>), and ZF_GAl4_KRAB (<partinfo>BBa_K2446037</partinfo>) (Fig 1).
  
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In this device, IL-6-scFv is a sensor for serum IL-6 level. The Notch domain undergoes two proteolytic cleavages upon the binding of ligands to the extracellular domain, which allows the release of the intracellular domain into the cytoplasm and the nucleus (Morsut et al., 2016). Gal4KRAB is a transcriptional inhibitor, where Gal4 binds to UAS (Gal4-binding region) in DNA and KRAB binds to the promoter attached to UAS, allowing inhibition of the downstream gene expression (Morsut et al., 2016; Witzgall et al., 1994).
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4175008 SequenceAndFeatures</partinfo>
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IL-6 is found to play a major role in the pathophysiology of cytokine release syndrome (CRS), the most common adverse effect during CAR-T therapy (Shimabukuro-Vornhagen et al., 2018; Xiao et al., 2021). The elevated IL-6 during CAR-T therapy is mostly released by monocyte/macrophage cell lines induced by target cell pyroptosis upon T cell killing (Norelli et al., 2018; Shimabukuro-Vornhagen et al., 2018). Therefore, we created this device to set a negative ‘switch’ for CAR expression in the case of high serum IL-6 level. We hoped that this would help prevent severe CRS.
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 +
We expected that when the concentration of IL-6 is high, the binding of IL-6 with anti-IL-6 scFv would cause the Notch core domain to undergo proteolytic cleavages (Fig 1). As a result, the Gal4-KRAB domain would be released into the nucleus. We intended to express anti-CD19 CAR with UAS-pSV40 as the promoter in the CAR-T cells. Therefore, the DNA binding domain of Gal4 would bind to the UAS region, enabling the KRAB domain to inhibit the SV40 promoter. In this way, the expression of CAR would be inhibited. As the cytotoxicity effect of CAR-T cells would be weaker, the target cells would undergo less pyroptosis. Consequently, fewer monocytes/macrophages would be induced to release IL-6. The serum level of IL-6 would thus decrease.
 +
 
 +
When the concentration of IL-6 is low, IL-6R would fail to bind with IL-6 due to its low affinity. As the promoter upstream of CAR, pSV40, is a strong promoter, the CAR expression would be constitutive. Ideally, these would collectively allow the CAR-T cells to exert its cytotoxic effect under low IL-6 concentration (Fig 1).
 +
 
 +
Hopefully, these would help maintain the IL-6 level in a normal range during CAR-T therapy.
 +
 
 +
===References===
 +
Morsut, L., Roybal, K.T., Xiong, X., Gordley, R.M., Coyle, S.M., Thomson, M., Lim, W.A., 2016. Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780–791. https://doi.org/10.1016/j.cell.2016.01.012
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Norelli, M., Camisa, B., Barbiera, G., Falcone, L., Purevdorj, A., Genua, M., Sanvito, F., Ponzoni, M., Doglioni, C., Cristofori, P., Traversari, C., Bordignon, C., Ciceri, F., Ostuni, R., Bonini, C., Casucci, M., Bondanza, A., 2018. Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells. Nat. Med. 24, 739–748. https://doi.org/10.1038/s41591-018-0036-4
 +
 
 +
Shimabukuro-Vornhagen, A., Gödel, P., Subklewe, M., Stemmler, H.J., Schlößer, H.A., Schlaak, M., Kochanek, M., Böll, B., von Bergwelt-Baildon, M.S., 2018. Cytokine release syndrome. J. Immunother. Cancer 6, 56. https://doi.org/10.1186/s40425-018-0343-9
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Witzgall, R., O’Leary, E., Leaf, A., Onaldi, D., Bonventre, J.V., 1994. The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proc. Natl. Acad. Sci. U. S. A. 91, 4514–4518. https://doi.org/10.1073/pnas.91.10.4514
  
 +
Xiao, X., Huang, S., Chen, S., Wang, Y., Sun, Q., Xu, X., Li, Y., 2021. Mechanisms of cytokine release syndrome and neurotoxicity of CAR T-cell therapy and associated prevention and management strategies. J. Exp. Clin. Cancer Res. CR 40, 367. https://doi.org/10.1186/s13046-021-02148-6
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 12:06, 10 October 2022


IL-6_scfv-Notch-Gal4KRAB

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 60
    Illegal XbaI site found at 271
    Illegal PstI site found at 55
    Illegal PstI site found at 1144
    Illegal PstI site found at 1375
    Illegal PstI site found at 1416
    Illegal PstI site found at 1462
    Illegal PstI site found at 2494
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 60
    Illegal PstI site found at 55
    Illegal PstI site found at 1144
    Illegal PstI site found at 1375
    Illegal PstI site found at 1416
    Illegal PstI site found at 1462
    Illegal PstI site found at 2494
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 60
    Illegal BglII site found at 1052
    Illegal BamHI site found at 76
    Illegal XhoI site found at 19
    Illegal XhoI site found at 1036
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 60
    Illegal XbaI site found at 271
    Illegal PstI site found at 55
    Illegal PstI site found at 1144
    Illegal PstI site found at 1375
    Illegal PstI site found at 1416
    Illegal PstI site found at 1462
    Illegal PstI site found at 2494
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 60
    Illegal XbaI site found at 271
    Illegal PstI site found at 55
    Illegal PstI site found at 1144
    Illegal PstI site found at 1375
    Illegal PstI site found at 1416
    Illegal PstI site found at 1462
    Illegal PstI site found at 2494
    Illegal NgoMIV site found at 1288
    Illegal NgoMIV site found at 2279
    Illegal NgoMIV site found at 2414
    Illegal AgeI site found at 1096
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Figure 1. The schematic of IL-6_scfv-Notch-Gal4KRAB under low and high serum IL-6 level.

This device incorporates anti-IL-6 scFv (BBa_K4175000), the Notch core domain (BBa_K4175001), and ZF_GAl4_KRAB (BBa_K2446037) (Fig 1).

In this device, IL-6-scFv is a sensor for serum IL-6 level. The Notch domain undergoes two proteolytic cleavages upon the binding of ligands to the extracellular domain, which allows the release of the intracellular domain into the cytoplasm and the nucleus (Morsut et al., 2016). Gal4KRAB is a transcriptional inhibitor, where Gal4 binds to UAS (Gal4-binding region) in DNA and KRAB binds to the promoter attached to UAS, allowing inhibition of the downstream gene expression (Morsut et al., 2016; Witzgall et al., 1994).

IL-6 is found to play a major role in the pathophysiology of cytokine release syndrome (CRS), the most common adverse effect during CAR-T therapy (Shimabukuro-Vornhagen et al., 2018; Xiao et al., 2021). The elevated IL-6 during CAR-T therapy is mostly released by monocyte/macrophage cell lines induced by target cell pyroptosis upon T cell killing (Norelli et al., 2018; Shimabukuro-Vornhagen et al., 2018). Therefore, we created this device to set a negative ‘switch’ for CAR expression in the case of high serum IL-6 level. We hoped that this would help prevent severe CRS.

We expected that when the concentration of IL-6 is high, the binding of IL-6 with anti-IL-6 scFv would cause the Notch core domain to undergo proteolytic cleavages (Fig 1). As a result, the Gal4-KRAB domain would be released into the nucleus. We intended to express anti-CD19 CAR with UAS-pSV40 as the promoter in the CAR-T cells. Therefore, the DNA binding domain of Gal4 would bind to the UAS region, enabling the KRAB domain to inhibit the SV40 promoter. In this way, the expression of CAR would be inhibited. As the cytotoxicity effect of CAR-T cells would be weaker, the target cells would undergo less pyroptosis. Consequently, fewer monocytes/macrophages would be induced to release IL-6. The serum level of IL-6 would thus decrease.

When the concentration of IL-6 is low, IL-6R would fail to bind with IL-6 due to its low affinity. As the promoter upstream of CAR, pSV40, is a strong promoter, the CAR expression would be constitutive. Ideally, these would collectively allow the CAR-T cells to exert its cytotoxic effect under low IL-6 concentration (Fig 1).

Hopefully, these would help maintain the IL-6 level in a normal range during CAR-T therapy.

References

Morsut, L., Roybal, K.T., Xiong, X., Gordley, R.M., Coyle, S.M., Thomson, M., Lim, W.A., 2016. Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780–791. https://doi.org/10.1016/j.cell.2016.01.012

Norelli, M., Camisa, B., Barbiera, G., Falcone, L., Purevdorj, A., Genua, M., Sanvito, F., Ponzoni, M., Doglioni, C., Cristofori, P., Traversari, C., Bordignon, C., Ciceri, F., Ostuni, R., Bonini, C., Casucci, M., Bondanza, A., 2018. Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells. Nat. Med. 24, 739–748. https://doi.org/10.1038/s41591-018-0036-4

Shimabukuro-Vornhagen, A., Gödel, P., Subklewe, M., Stemmler, H.J., Schlößer, H.A., Schlaak, M., Kochanek, M., Böll, B., von Bergwelt-Baildon, M.S., 2018. Cytokine release syndrome. J. Immunother. Cancer 6, 56. https://doi.org/10.1186/s40425-018-0343-9

Witzgall, R., O’Leary, E., Leaf, A., Onaldi, D., Bonventre, J.V., 1994. The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proc. Natl. Acad. Sci. U. S. A. 91, 4514–4518. https://doi.org/10.1073/pnas.91.10.4514

Xiao, X., Huang, S., Chen, S., Wang, Y., Sun, Q., Xu, X., Li, Y., 2021. Mechanisms of cytokine release syndrome and neurotoxicity of CAR T-cell therapy and associated prevention and management strategies. J. Exp. Clin. Cancer Res. CR 40, 367. https://doi.org/10.1186/s13046-021-02148-6