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Design

Module1

We notice that specific endogenous transcription factors (TFs) influence cancer cells almost every aspects, so we want to utilize this TFs as synthetic promotors to initiate therapy circuit accurately towards TNBC cells (1) Also, at the same time of improving the accuracy of therapy , designing synthetic promoter activated in TNBC specifically can lower the risk of cancer therapy, especially lower the system Toxicity comparing to conventional chemotherapy, because normal cells mostly have too little endogenous TFs to initiate our therapy circuit. Though, there are few cases that some normal cells have enough endogenous TFs to initiate the circuit. - In our design, Module1 performs as sensor to receive the activation of endogenous TF , also as trigger to initiate the downstream Module. Considering these, we add sequence of synthetic TF——GAD to activate downstream elements.(2)

In TNBC cells, GATA3 as endogenous transcription factor, specifically high expresses so that synthetic promoter(s(GATA3)p) can largely be activated and express a great deal of GAD.

In most cases, normal cells hardly express GAD ,though they are transformed to have the same synthetic promoter as the left picture, because the normal cells lack endogenous TF as GATA3 ,only little amount of GATA3 seldom initiate to express GAD.

Thus, we utilize specifically abundant endogenous TF as the limitation to initiate expression of GAD.

While one problem comes, though normal cells have little GATA3, there are still few cases that GATA3 can binding to the synthetic promoter we design to express GAD wrongly.

We notice that some endogenous miRNAs can take on low level in various cancer cells(3). Considering these risks, we utilize endogenous miRNA as another limitation to make the initiation more specifically and lower the potential accidentally injure.

We screen some endogenous miRNA in TNBC cells and take on specifically lower level, while in normal cells, the miRNA takes on higher level, like miR101 showed in left picture(4).

So we add the binding sites of the miRNA (like miR101-BS) to make the level of miRNA as the limitation to initiate our circuit: normal cells and the few TNBC cells with high level of miR101 can’t initiate GAD because excess miR101 will bind to miR101-BS so that translation won’t finish.

In normal cells, mostly take on high expression of the miRNA and lack of endogenous TFs , make them harder to initiate GAD, so the accidently initiation is very little.

While few cases of TNBC cells can express high level of the chosen miRNA, then the miRNA-BS will bind miRNA and impede the expression of GAD in Module1.

To handle all the types of TNBC cells, we should control the level of miRNA level and prevent miRNA/miRNA-BS from binding in TNBC cells.

Module2

To initiate Module1 under high level of miRNA present or remaining miRNA leak in TNBC cells , we design a new module named Module2 which contains highly specific synthetic promoter s(ESR1)p , serial sequences of Sponge and EGFP(2)(5):

The promotor of Module2 ,s(ESR1)p,(2) is a synthetic promoter which is activated by ESR1——endogenous TF in TNBC cells. Thus, almost all TNBC cells can initiate Module2. When the expression of chosen miRNA takes on too high, Module2 can express Sponge and bind the excess of the miRNA to make sure the level of miRNA can reduce to the stage that won’t make miRNA/miRNA-BS bind together

In normal cells, lacking of endogenous ESR1 makes Module2 not initiate so that none of Sponge is expressed, the level of miRNA in normal cells still maintains high level.

Extremely few cases in normal cells may initiate Module2 because ESR1 initiates Module2 and express Sponge

Module3

The trigger of Module3 relies on synthetic promoter by design, since Module1 can express GAD, we synthesize the promoter——G8p to receive the activation of GAD(2)

To make sure the whole circuit can specifically work , we add miR-BS to control the low level of miRNA in TNBC cells, also to strength the prevention of accidentally injure in normal cells.

Module3 is highly modularized , we can place sequences of anti-tumor effector like yCD between G8p and miR-BS.

The anti-tumor effector we choose is adding the sequence of yCD , we expect that yCD will mildly convert the nontoxic prodrug 5-FC to the conventional chemotherapy drug 5-FU (6)

5-FU is the finally product to kill TNBC cells, its cytotoxic occurs following its conversion to 5-FdUMP, while 5-FdUMP is an irreversible inhibitor of thymidylate synthase by dTTP deprivation and causes DNA strand breakage, leading to cell death

Through placing this suicide gene therapy approached like yCD, we lower the system toxicity and choose stable but widely used drug 5-FU to achieve broad spectrum killing quantificational towards TNBC cells by enzymic reaction.

Advanced assumptions

To show our modularized circuit, we demonstrate one example to certify

Module3 is highly modularized by replacing the sequences between G8p and miR-BS, as the figure showed, we add HIF-1α ODDD to the N terminal of yCD.

Then, we add one limitation of environment condition——hypoxia , to more specifically initiate the whole circuit, may make our therapy circuit work more efficient towards some status of TNBC under hypoxia, since the development and invasion are close to the status of O2 intake.

References

[1] R. Li, J. Campos, and J. Iida. A Gene Regulatory Program in Human Breast Cancer. Genetics, Vol. 201, 1341–1348 December 2015.

[2] Nissim et al. Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy. Cell 171, 2017. 1138–1150.

[3] Pannapa Pinweha et al. MicroRNAs and oncogenic transcriptional regulatory networks controlling metabolic reprogramming in cancers Computational and Structural Biotechnology Journal 14 (2016) 223–233 http://dx.doi.org/10.1016/j.csbj.2016.05.005.

[4] Haitao guan, Zhijun dai, Yuguang ma, Zhongwei wang, Xiaoxu liu and Xijing wang.Microrna-101 inhibits cell proliferation and induces apoptosis by targeting EYA1 in breast cancer . INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE .37: 1643-1651, 2016.

[5]Tomas Barta, Lucie Peskova & Ales Hampl. miRNAsong: a web-based tool for generation and testing of miRNA sponge constructs in silico Scientific Reports | 6:36625 |

[6]Tiana D. Warren, Krishna Patel, James R. Eshleman, and Marc Ostermeier. Protein-Programmed Accumulation of Yeast Cytosine Deaminase in Cancer Cells in Response to Mock-Hypoxia ACS Synth. Biol. April 18, 2019.

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