Part:BBa_K4839022
TRE3GS promoter- Anti-GFP BioPROTAC
The part consists of promoter PGK, downstream rtTA and promoter TRE3GS,downstream Anti-GFP BioPROTAC. Among them, rtTA is a trans-activator in the Tet-inducible transcription system (Tet-ON system) that activates pTRE. rtTA is inactive on its own, and the complex formed by the addition of Dox selectively binds to pTRE and initiates downstream transcription. Downstream Anti-GFP BioPROTAC encodes the vhhGFP4-SPOP chimera. This chimera recognizes and binds H2B-GFP and induces its ubiquitinated degradation.
Figure1. Map for pLVX-TREG3S-FLAG-vhhGFP4-SPOP-TetOne-Puro.
We co-transfected 1 μg of pLVX-TREG3S-FLAG-vhhGFP4-SPOP-TetOne-Puro and pCMV-H2B-eGFP-Neo into HEK293T cells seeded in a 12-well plate. We induced anti-GFP BioPROTAC expression by adding 2 μg of doxycycline for 48 hours. Microscopic examination showed (Figure 2a) that H2B-GFP was fully degraded after induction with doxycycline. We also performed further analysis using flow cytometry, which also showed significant GFP degradation (Figure 2b). We further analyzed the data generated from flow cytometry, and we can also observe from Figure 1d that GFP was efficiently degraded in the group co-transfected with pLVX-TREG3S-FLAG-vhhGFP4-SPOP-TetOne-Puro and induced with doxycycline.
Figure2. Proof of function of Tet-on bioPROTAC system. (a) Fluorescence microscopy images at a scale of 100 microns. (b) Flow cytometry result: left represent HEK293T transfected with two plasmids without doxyxycline; right represent HEK293T transfected with two plasmids with doxyxycline. (c) "D" represents doxycycline; "G" represents pCMV-H2B-eGFP-Neo, "P" represents pLVX-TREG3S-FLAG-vhhGFP4-SPOP-TetOne-Puro. (d) Bar plot representing different group's GFP rates after doxycycline induction.
We found that doxycycline induction can achieve a very good GFP degradation effect. Therefore, we wanted to further evaluate the system at the cellular level. Moreover, as H2B-GFP is a nuclear protein, it can also be degraded by BioPROTAC, which provides a good basis for our subsequent experiments on degrading IRF4/IRF5. We then set different doxycycline concentrations (100 ng/mL to 2000 ng/mL) to find the optimal induction concentration. Similarly, we obtained the following results through flow cytometry analysis (Figure 3). We found that the best protein degradation effect was achieved at a doxycycline concentration of 1000 ng/mL.
Figure3. Doxycycline gradient for induction of GFP degradation. (a) The horizontal axis represents the intensity of GFP, with stronger intensity towards the right side. (b) The dot plot shows the percentage of GFP at different doxycycline concentrations
The overall design of our project
In our design, the engineered macrophages, SYN-MACRO could recognize GPC3 via SNIPR. Through Notch signaling pathway, SNIPR will be cleaved 3 times by γ-secretase and release transcription factor Gal4VP64 to activate AntiIRF4-BioPROTAC expression, effectively degrading the key protein IRF4 that regulates macrophage M2 polarization. Thereby promoting macrophage M1 polarization (Figure 4). Further SYN-MACRO will release pro-inflammatory cytokines and recruit CD8 T cells for effective tumor eradication.
Figure 4. GPC3 induced M1 polarization by activation of anti-IRF4 BioPROTAC.
After effective tumor eradication, doxycycline is used to induce downstream AntiIRF5-BioPROTAC expression to restore the anti-inflammatory phenotype in the tumor microenvironment also as a safety module, while inhibiting chronic inflammation caused by triglyceride accumulation in liver tissues of many HCC patients, thereby reducing further damage to liver tissue caused by chronic inflammation, also for inhibition of M1 over-polarization (Figure 5)
Figure 5. Doxycycline induced M1 polarization by activation of anti-IRF5 BioPROTAC.
Figure 6. Schematic of the SYN-MACRO M1/M2 polarization.
So now we have the complete gene circuit design of SYN-MACRO (Figure 6). We can control the polarization process of SYN-MACRO in vivo. We hope to precisely target GPC3-positive liver cancer cells with SYN-MACRO and effectively polarize them towards the M1 phenotype, thereby restructuring the tumor microenvironment, promoting the occurrence of inflammation, and inhibiting the development of tumors.
The Anti-GFP BioPROTAC part act as a critical role at the begining of our concept. You may try to test the bioPROTAC system in your cell with this part and the part BBa_K4839021.
Reference
[1] Rothbauer, U. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3, 887–889 (2006).
[2] Shin, Y. J. et al. Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins. Sci Rep 5, 14269 (2015).
[3] Shen, H. et al. MDM2-Mediated Ubiquitination of Angiotensin-Converting Enzyme 2 Contributes to the Development of Pulmonary Arterial Hypertension. Circulation 142, 1190–1204 (2020).
[4] Klichinsky, M. et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 38, 947–953 (2020).
Sequence and Features
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Illegal PstI site found at 1897 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 257
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- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1250
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Illegal PstI site found at 1897 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1250
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Illegal NgoMIV site found at 2365
Illegal AgeI site found at 2174 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 99
Illegal BsaI.rc site found at 2746
Illegal SapI.rc site found at 2760
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