Difference between revisions of "Part:BBa K4175010"

 
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<partinfo>BBa_K4175010 short</partinfo>
 
<partinfo>BBa_K4175010 short</partinfo>
  
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When interleukin-6 (IL-6) concentration is high to an extent, IL-6 will bind to IL-6 receptor (IL-6R) (BBa_K4175002). Then, the Notch core domain (BBa_K4175001) would undergo intracellular proteolytic cleavage. As a result, the Gal4KRAB domain (BBa_K2446037) will be released into the cytosol and bound to the UAS-pSV40 (BBa_K4040033) in the nucleus. The expression of gene controlled by pSV40 promoter will thus be inhibited.
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===Sequence and Features===
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<partinfo>BBa_K4175010 SequenceAndFeatures</partinfo>
  
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===Usage and Biology===
 
===Usage and Biology===
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[[File:IL6R-Notch-Gal4KRAB.png|thumb|left|400px|<b>Figure 1.</b> The schematic of IL6R-Notch-Gal4KRAB under low and high serum IL-6 levels.]]
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This device is comprised of extracellular human IL-6R (aa 1-309) (<partinfo>BBa_K4175013</partinfo>), the Notch core domain (<partinfo>BBa_K4175001</partinfo>), and ZF_GAl4_KRAB (<partinfo>BBa_K2446037</partinfo>) (Fig 1).
  
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The usage and rationale of the synthetic device is quite similar to IL-6_scfv-Notch-Gal4KRAB (<partinfo>BBa_K4175008</partinfo>). Briefly speaking, we used IL-6R-Notch-Gal4KRAB as a negative ‘switch’ for CAR expression when the serum IL-6 level goes beyond normal. We hoped that implementation of this device to CAR-T cells would prevent severe cytokine release syndrome (CRS) from happening during CAR-T therapy, as IL-6 is a major culprit of CRS (Shimabukuro-Vornhagen et al., 2018). When the serum IL-6 level goes high, we expected that binding of IL-6 to IL-6R would trigger the proteolytic cleavage of Notch (Fig 1). Then the intracellular Gal4KRAB would be released into the nucleus. As we transfected the T cells with UAS-pSV40-aCD19-CAR (<partinfo>BBa_K4175008</partinfo>), the Gal4KRAB would then bind to the UAS region and inhibit pSV40 promoter. Consequently, the anti-CD19 CAR expression would be suppressed. The cytotoxic effect of CAR-T would be paused, and the monocytes/macrophages would temporarily stop producing more IL-6. Conversely, when the IL-6 level is low, IL-6R would not transduce signals due to its low affinity for IL-6 and the CAR would be constitutively expressed due to the strong promoter, SV40 (Fig 1). We hoped this would maintain the serum IL-6 level in a normal range.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4175010 SequenceAndFeatures</partinfo>
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The only difference between this part and Part:<partinfo>BBa_K4175008</partinfo> lies in the IL-6 sensor. For BBa_K4175008, we used anti-IL6 scFv as the IL-6 sensor; while for this part, we used extracellular IL-6R as the sensor. These two sensors may have different affinities for IL-6, which means that the threshold for these two devices to exert their suppressive effect on CAR expression might be different.
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For more details of usage and biology, please visit the part page of IL-6_scfv-Notch-Gal4KRAB (<partinfo>BBa_K4175008</partinfo>).
  
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===References===
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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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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K4175010 parameters</partinfo>
 
<partinfo>BBa_K4175010 parameters</partinfo>
 
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Revision as of 15:10, 10 October 2022


IL-6R-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 1117
    Illegal PstI site found at 1294
    Illegal PstI site found at 1519
    Illegal PstI site found at 1560
    Illegal PstI site found at 1606
    Illegal PstI site found at 2638
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 60
    Illegal PstI site found at 55
    Illegal PstI site found at 1117
    Illegal PstI site found at 1294
    Illegal PstI site found at 1519
    Illegal PstI site found at 1560
    Illegal PstI site found at 1606
    Illegal PstI site found at 2638
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 60
    Illegal BamHI site found at 76
    Illegal BamHI site found at 1194
    Illegal XhoI site found at 19
  • 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 1117
    Illegal PstI site found at 1294
    Illegal PstI site found at 1519
    Illegal PstI site found at 1560
    Illegal PstI site found at 1606
    Illegal PstI site found at 2638
  • 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 1117
    Illegal PstI site found at 1294
    Illegal PstI site found at 1519
    Illegal PstI site found at 1560
    Illegal PstI site found at 1606
    Illegal PstI site found at 2638
    Illegal NgoMIV site found at 1432
    Illegal NgoMIV site found at 2423
    Illegal NgoMIV site found at 2558
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Figure 1. The schematic of IL6R-Notch-Gal4KRAB under low and high serum IL-6 levels.

This device is comprised of extracellular human IL-6R (aa 1-309) (BBa_K4175013), the Notch core domain (BBa_K4175001), and ZF_GAl4_KRAB (BBa_K2446037) (Fig 1).

The usage and rationale of the synthetic device is quite similar to IL-6_scfv-Notch-Gal4KRAB (BBa_K4175008). Briefly speaking, we used IL-6R-Notch-Gal4KRAB as a negative ‘switch’ for CAR expression when the serum IL-6 level goes beyond normal. We hoped that implementation of this device to CAR-T cells would prevent severe cytokine release syndrome (CRS) from happening during CAR-T therapy, as IL-6 is a major culprit of CRS (Shimabukuro-Vornhagen et al., 2018). When the serum IL-6 level goes high, we expected that binding of IL-6 to IL-6R would trigger the proteolytic cleavage of Notch (Fig 1). Then the intracellular Gal4KRAB would be released into the nucleus. As we transfected the T cells with UAS-pSV40-aCD19-CAR (BBa_K4175008), the Gal4KRAB would then bind to the UAS region and inhibit pSV40 promoter. Consequently, the anti-CD19 CAR expression would be suppressed. The cytotoxic effect of CAR-T would be paused, and the monocytes/macrophages would temporarily stop producing more IL-6. Conversely, when the IL-6 level is low, IL-6R would not transduce signals due to its low affinity for IL-6 and the CAR would be constitutively expressed due to the strong promoter, SV40 (Fig 1). We hoped this would maintain the serum IL-6 level in a normal range.

The only difference between this part and Part:BBa_K4175008 lies in the IL-6 sensor. For BBa_K4175008, we used anti-IL6 scFv as the IL-6 sensor; while for this part, we used extracellular IL-6R as the sensor. These two sensors may have different affinities for IL-6, which means that the threshold for these two devices to exert their suppressive effect on CAR expression might be different.

For more details of usage and biology, please visit the part page of IL-6_scfv-Notch-Gal4KRAB (BBa_K4175008).

References

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