Part:BBa_K4839020

Anti-GPC3 SNIPR

In our design, we construct anti-GPC3 SNIPR and assemble it to our synthetic macrophage, which is called SYN-MACRO (See design). We wish our artificially design macrophage can specifically recognize GPC3-positive cancer cells (such as Huh7 and THP-1). While SYN-MACRO specifically target to the GPC3 receptor, it will automatically mediate the downstream signal transduction and transform itself from M2 phenotype to M1 phenotype. The M1 and M2 phenotype is associate with the proinflammatory anti-inflammatory of the macrophage. The M1 phenotype will promote inflammatory response and thus prevent the tumor progression. (Figure1.)

Figure1. The overview of SYN-MACRO

So how can SYN-MACRO recognize GPC3 receptor and activate the downstream gene expression? We use a synthetic receptor called SNIPR (synthetic intramembrane proteolysis receptors) to solve this problem. (Fugure2.)

Figure2. The structure of SNIPR

SNIPR is a highly customizable synthetic biology component based on the Notch signaling pathway. Notch is a type 1 transmembrane protein that is activated by regulated intramembrane proteolysis (RIP). As shown in Figure 3, the activation process is a continuous reaction involving the shedding of disintegrin and metalloprotease (ADAM) mediated extracellular domain (ECD), cleavage of the transmembrane domain (TMD) mediated by γ-secretase, and release of the transcription factor (TF) into the cytoplasm, which is then transported to the nucleus. (Figure3.)

Figure3. Activation process of the Notch signaling pathway

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 159
  Illegal PstI site found at 241
  Illegal PstI site found at 987
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 159
  Illegal PstI site found at 241
  Illegal PstI site found at 987
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 159
  Illegal BamHI site found at 1498
  Illegal BamHI site found at 1537
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 159
  Illegal PstI site found at 241
  Illegal PstI site found at 987
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 159
  Illegal PstI site found at 241
  Illegal PstI site found at 987
  
• 1000
  COMPATIBLE WITH RFC[1000]