Difference between revisions of "Part:BBa K4839020"

Latest revision as of 15:53, 12 October 2023

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. (Figure 1.)

Figure 1. 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. (Fugure 2.)

Figure 2. The structure of SNIPR and Sanger sequencing results for successful construction.

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. (Figure 3.)

Figure 3. Activation process of the Notch signaling pathway

As for our experiment, firstly, we transfected pLVX-CMV-Anti-GPC3_SNIPR-PGK-Puro and pHR-Gal4UAS-eGFP-PGK-mcherry-BleoR into HEK293T cells using Mikx superluminal high-efficiency transfection reagent. We identified the expression of the two proteins by Western blot and fluorescence microscopy (Figure 4). We observed clear bands of Anti-GPC3_SNIPR and bright mCherry fluorescence. At the same time, we also found no significant leakage expression of GFP, which first proved that the basic functions of each module of the system were normal.

Figure 4. Expression validation. (a) Western blot result for the expression of Anti-GPC3_SNIPR in HEK293T. (b) Fluorescence microscopy result for the expression of mcherry.

After completing the verification of the separate expression of the two proteins, we needed to find a liver cancer cell line with high expression of GPC3. Through literature search, we found Huh7 as a co-culture object with the cells transfected with SNIPR and the reporter system. We attempted to transfect pLVX-CMV-Anti-GPC3_SNIPR-PGK-Puro and pHR-Gal4UAS-eGFP-PGK-mcherry-BleoR into HEK293T cells at 12-hour intervals. We found a small amount of GFP leakage expression under the fluorescence microscope, which we speculated might be due to self-cleavage of a small amount of SNIPR, a small amount of transcription factor entering the nucleus, or a small amount of pHR-Gal4UAS-eGFP-PGK-mcherry-BleoR binding to Gal4VP64 before it was transferred into nucleus, leading to a small amount of GFP leakage. Next, we co-cultured HEK293T and Huh7 cells after transfecting the first plasmid for 24 hours and observed the fluorescence results after another 24 hours (Figure 5). We found a significant increase in GFP expression in the co-culture groups, while the GFP expression level was significantly weaker in the groups without SNIPR receptor transfection after co-culture.

Figure 5. Transfected HEK293T Co-cultured with Huh7 present an increase in GFP expression.

We wanted to better present the experimental results through quantitative analysis, so we used flow cytometry to better detect the level of green fluorescence, which reflects the function of SNIPR recognizing GPC3 and activating downstream gene expression. As shown in Figure 1-4a,b, we can clearly see that transfected HEK293T cells showed a strong increase in green fluorescence expression after co-culture. We also performed statistical analysis on the experimental results. We used the ratio of green fluorescence cells as the numerator and the sum of the ratio of red fluorescence and green fluorescence as the denominator to measure the ability of SNIPR to recognize and activate downstream gene expression. We found a very significant increase in GFP expression in the co-culture group. Secondly, we found that there was a significant difference in whether to first transfect pLVX-CMV-Anti-GPC3_SNIPR-PGK-Puro or pHR-Gal4UAS-eGFP-PGK-mcherry-BleoR. For the group that was transfected with pHR-Gal4UAS-eGFP-PGK-mcherry-BleoR first, the mCherry expression intensity was higher, but the difference in green fluorescence level between the co-culture and non-co-culture groups was smaller. For the group that was transfected with pLVX-CMV-Anti-GPC3_SNIPR-PGK-Puro first, the mCherry expression intensity was lower, but the difference in green fluorescence level between the co-culture and non-co-culture groups was greater. Since gene expression requires a certain amount of time, the experimental results also demonstrate that anti-GPC3 SNIPR has the function we expected.

Figure 6. Quantitative analysis for function validation of anti-GPC3 SNIPR in HEK293T. (a) Flow cytometry result for group without co-culture. (b) Flow cytometry result for co-culture group. (c) Bar 1&2: only transfected with pHR-Gal4UAS-eGFP-PGK-mcherry-BleoR; Bar 3&4: transfected with pHR-Gal4UAS-eGFP-PGK-mcherry-BleoR at 0h and pLVX-CMV-Anti-GPC3_SNIPR-PGK-Puro at 12h; Bar 5&6: transfected with pLVX-CMV-Anti-GPC3_SNIPR-PGK-Puro at 0h and pHR-Gal4UAS-eGFP-PGK-mcherry-BleoR at 12h. Data is expressed as means ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001; NS, not significant,(two-tailed Student’s t-test)

Therefore, we wanted to validate the function of anti-GPC3 SNIPR in the commonly used monocyte cell line THP-1 for macrophage research . We have discussed in detail the methods for transfecting/infecting THP-1 in the Engineering section. In the end, we used the second-generation lentivirus system (which we also thoroughly evaluated for safety, submitted a check-in form to HQ and got approval before the experiment began) as the carrier for infecting THP-1, and achieved some success. We used HEK293T cells for lentivirus packaging, added the collected virus solution to THP-1 culture medium, induced macrophage differentiation with PMA 48h later, and co-cultured with Huh7 at a 1:1 ratio 24h later. Fluorescence microscopy was performed every 24h to determine if SNIPR could effectively recognize GPC3 in THP-1. At 24h after co-culture, we successfully observed green fluorescence emitted by THP-1 cells in the co-culture group (Figure 7). Due to the low transduction efficiency of lentivirus, we could only observe limited fluorescence levels and did not have the conditions for quantitative analysis. However, we were still able to observe significant green fluorescence in the co-culture group transfected with both pLVX-CMV-Anti-GPC3_SNIPR-PGK-Puro and pHR-Gal4UAS-eGFP-PGK-mcherry-BleoR, while no green fluorescence was observed in the co-culture group transfected with only pHR-Gal4UAS-eGFP-PGK-mcherry-BleoR.

Figure 7. Function validation of anti-GPC3 SNIPR in PMA-induced THP-1. Transduced THP-1 cells with lentivirus carrying pHR-UAS-GFP-PGK-mcherry and pLVX-CMV-SNIPR are shown at a scale of 10 microns to better observe the green fluorescence. Transduced THP-1 cells with lentivirus carrying pHR-UAS-GFP-PGK-mcherry are shown at a scale of 100 microns, demonstrating the inability to observe green fluorescence while still being able to see the red fluorescence

We also conducted further investigations into the relationship between transfection of varying amounts of pLVX-CMV-Anti-GPC3_SNIPR-PGK-Puro and enhanced green fluorescence in HEK293T cells. We transfected gradient amounts (0-2000ng) of pLVX-CMV-Anti-GPC3_SNIPR-PGK-Puro to each well of a 12-well plate at 0h. At 12h, we transfected each well with 1000ng of pHR-UAS-GFP-PGK-mcherry. After 24h, we co-cultured with Huh7 at a 1:1 ratio. Flow cytometry was performed 24h later for quantitative analysis. The results are shown in Figure 1-6. We observed that as the amount of pLVX-CMV-Anti-GPC3_SNIPR-PGK-Puro transfection increased, the leakage of green fluorescence also increased, but there was still a significant difference in green fluorescence expression between the co-culture and non-co-culture groups in each group. Based on these results, we believe that transfection with 500ng of pLVX-CMV-Anti-GPC3_SNIPR-PGK-Puro can control GFP leakage expression to a certain extent while also demonstrating the function of anti-GPC3 SNIPR well.

Figure 8. Investigation of the relationship between varying amounts of pLVX-CMV-Anti-GPC3_SNIPR-PGK-Puro and enhanced green fluorescence in HEK293T cells. Data is expressed as means ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001; NS, not significant, (two-tailed Student’s t-test).

The overall design of our SNIPR and the downstream gene in 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 9). Further SYN-MACRO will release pro-inflammatory cytokines and recruit CD8 T cells for effective tumor eradication.

Figure 9. 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 10)

Figure 10. Doxycycline induced M1 polarization by activation of anti-IRF5 BioPROTAC.

Figure 11. Schematic of the SYN-MACRO M1/M2 polarization.

So now we have the complete gene circuit design of SYN-MACRO (Figure 11). 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] Zhu, I. et al. Modular design of synthetic receptors for programmed gene regulation in cell therapies. Cell 185, 1431-1443.e16 (2022).

[2] Morsut, L. et al. Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780–791 (2016).

[3] Sun, C. K., Chua, M.-S., He, J. & So, S. K. Suppression of Glypican 3 Inhibits Growth of Hepatocellular Carcinoma Cells through Up-Regulation of TGF-β2. Neoplasia 13, 735–747 (2011).

[4] Chanput, W., Mes, J. J. & Wichers, H. J. THP-1 cell line: an in vitro cell model for immune modulation approach. Int Immunopharmacol 23, 37–45 (2014).

[5] Genin, M., Clement, F., Fattaccioli, A., Raes, M. & Michiels, C. M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide. BMC Cancer 15, 577 (2015).

[6] Saerens, D. et al. Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J Mol Biol 352, 597–607 (2005).

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