Team:LZU-CHINA/Design

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Freeze! Pancreatic cancer!

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Design

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DELIVERY INHIBITOR KILLER

DELIVERY

Delivery system

We extract TIL cells from patients’ cancer cells and transfect with exosome booster and miRNA module, then we inject the engineered TIL cells into patients’ body. The TIL cells can target tumor mass where cancer cells locate and come into play. Cancer cells’ ability of uptaking exosomes is greatly increased in tumor microenvironment due to an acidic environment. Also, cancers have type-specific mutations or protein alterations that result in increased exosomal uptake.

When TIL cells get close to cancer cells, they sense the hypoxia condition and HIF-1 are produced, they combine HRE (hypoxia response elements, we quintuple HRE to ensure the robustness of promoters) and then activate minimized CMV promoter, thus promoting the expression of exosomes.

By adding different inducers, TIL cells will start expressing different concentrations of miRNA. Exosomes can encapsule miRNA automatically and send them to cancer cells. Cancer cell can uptake them at an increased speed. By adding different combination of inducers, we can explore whether the summary effect of miRNA is better than the effect of single miRNA, thus finding out the best way to suppress tumor activity.

Fig

INHIBITOR

Remote transfer inhibition system

Many studies have confirmed that tumorigenesis is associated with uncontrolled cell proliferation and apoptosis. The constitutive activation of the MAPK signaling pathway occurs in many tumor cell lines (pancreatic cancer, colon cancer, lung cancer, ovarian cancer, and kidney cancer) and primary tumors (kidney cancer, colon cancer, and lung cancer) of various human organs.

We edited the RAS/RAF gene in engineered TIL cells to ligature the RAS transcribed to the N-TEVP, and the RAF to the C-TEVP. The exosomes deliver mRNA to the cells of interest. Once the target cell is cancerous, the RAS-RAF-MAPK pathway is activated in large amounts, and active TEVP is produced.

Fig

In pancreatic cancer, ALKBH5 is under-represented, inhibition of the wnt pathway is weakened, and pathway activation. We inserted a TEVP target site between N-ALKBH5 and C-ALKBH5. After activation of RAS-RAF, active TEVP is produced, and the target sequence between ALKBH5 is cleaved by TEVP to synthesize an active protein. ALKBH5 utilizes demethylase activity to increase the expression of APC in the 3'-UTR region of the tumor suppressor gene APC messenger, up-regulating the expression of APC in the downstream of the wnt pathway, and reversing the low expression of ALKBH5 on the pancreas. The promotion of cancer proliferation and invasion and metastasis.

KILLER

Invasive cancer cell killing system

The study found that in the tumor tissues of pancreatic cancer, tumor cells accounted for less than 30%, more inflammatory interstitial cells and fibrous tissue, which makes the pancreatic cancer have the characteristics of hypoxia and hypoxia. Previous studies have found that HIF-1 is a key link in the development of pancreatic cancer. The positive rate of HIF-1 in pancreatic cancer tissues was as high as 88%, while that in normal tissues was only 16%. When hypoxia is localized, HIF-1 is overexpressed and promotes the progression of pancreatic cancer in a variety of ways.

We stabilized the gene into engineered TIL cells and ligated the TVMVP gene after the HIF-1 hypoxia-inducible promoter. Once the rapid increase in cancer cells leads to an increase in cellular oxygen consumption, resulting in an anoxic microenvironment, the hypoxia-inducible promoter will be activated, and the mRNA of TVMVP will be transcribed and transported to the target cells by exosomes.

Caspase-3 is a protease that is an important component of CTL cell killing mechanism. We achieved a regulatable cell killing effect by transforming caspase3 in TCL cells. We linked the target site of TEVP between C-casp3 and N-casp3, and the TVMVP target site was ligated between N-casp3 and the efferent signal peptide.

In the delivery of mRNA to the target cell by exosomes, cleaving by TEVP produces active casp3 (but it is still linked to the signal peptide, which cannot remain in the cell); when subjected to tumor hypoxia The influence of microenvironment causes a large amount of expression of TVMVP, which cleaves the junction of casp3 and the efferent signal peptide, so that the active casp3 remains in the target cell to exert a cell killing effect.

Fig

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