Difference between revisions of "Part:BBa K4839023"

Latest revision as of 15:25, 12 October 2023

TRE3GS promoter- Anti- IRF4 BioPROTAC

This component consists of promoter PGK, downstream rtTA, and promoter TRE3GS, downstream Anti-IRF4 BioPROTAC. rtTA acts as a trans-activator in the Tet-inducible transcription system (Tet-ON system) and binds to the exogenous Dox to activate pTRE, initiating downstream transcription, and the downstream Anti-IRF4 BioPROTAC encoded product recognizes and binds IRF4 and induces its ubiquitinated degradation.

We have successfully constructed the IRF4 Binding protein (anti-IRF4 BioPROTAC) and assembled it into the pLVX-TREG3S-FLAG-TetOne-Puro vector regulated by doxycycline-controlled Tet-on system. We have also completed the construction of pLVX-TREG3S-FLAG-PU.1-(SSG)3-SPOP-TetOne-Puro and the sequencing results are shown in Figure 2.

Figure2. Sequencing results confirm the successful construction of IRF4 Binding protein (PU.1-SPOP).

The C-terminal E3 adaptor/E3 domain was selected based on the suggestion of Professor Ting Pan from Sun Yat-sen University School of Medicine. We chose SPOP, which has been widely used in previous studies, instead of using the search results from the database. We then designed the downstream experimental plan (Figure 1), aiming to induce the expression of anti-IRF4 BioPROTAC using doxycycline and achieve degradation of IRF4.

Figure1.A schematic proof of function for controllable degradation of IRF4 protein

we transfected HEK293T cells in a 12-well plate with 1 μg of pLVX-TREG3S-FLAG-PU.1-(SSG)3-SPOP-TetOne-Puro and 1 μg of pcDNA3.1-CMV-FLAG-IRF4/pcDNA3.1-CMV-HA-IRF4, induced expression with 1 μg/mL of doxycycline for 48h, and collected the protein for western blot analysis. The results are shown in Figure 2, where we observed significant degradation of both HA-IRF4 and FLAG-IRF4.

Figure2. Western blot signing degrdation of IRF4. "D" represents doxycycline.

Due to time constraints, we have not been able to conduct this experiment in the THP-1 cell line. However, in the HEK293T cell experiment, we have observed a certain degree of IRF4 degradation, which is a breakthrough that has not been achieved in previous research. We are very excited about these results. We will then continue to optimize the transfection/infection efficiency for THP-1 cells and have also contacted Professor Chong Wu, a macrophage expert from Sun Yat-sen University School of Life Sciences, to explore ways to optimize related experiments for THP-1 cells. Additionally, we will conduct further experimental validation for the designed anti-IRF5 BioPROTAC and optimize anti-IRF4 BioPROTAC using bioinformatics methods to enhance its degradation efficiency.

The overall design of this part

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 3). Further SYN-MACRO will release pro-inflammatory cytokines and recruit CD8 T cells for effective tumor eradication.

Figure 3. 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 4)

Figure 4. Doxycycline induced M1 polarization by activation of anti-IRF5 BioPROTAC.

Figure 5. Schematic of the SYN-MACRO M1/M2 polarization.

So now we have the complete gene circuit design of SYN-MACRO (Figure 5). 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.

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