Description graphic

Project Description

ikona 1

Utilizes mRNA technology with toehold switches to induce pyroptosis in liver cancer cells, triggering an antitumor immune response.

ikona 2

Analyzes transcriptomic data from tumor and healthy cell biopsies to generate personalized toehold switch sequences tailored to the patient’s mutations.

ikona 3

Detects liver cancer–specific AFP RNA sequences to ensure precise therapeutic action exclusively within target tissues.

Project motivation

The initial goal of our project was liver cirrhosis, which in Poland is a major problem compared to other European countries. While studying this subject, we realized that the most severe complication of liver cirrhosis, and the one for which medicine currently has no effective answer, is hepatocellular carcinoma (HCC).

We are based in Warsaw, at one of the largest liver transplantation centers in Europe – the University Clinical Center of the Medical University of Warsaw. This unique center performs more than 300 liver transplants annually, and combines the Clinical Oncology Department with the Radiology Clinic, allowing HCC patients to receive coordinated care. Sadly, it is the only such comprehensive center in Poland. Many patients in other hospitals face long waiting times, and during this period their disease may progress. Once progression occurs, they are often referred to other treatment options unavailable in their original center. We therefore decided to develop a therapy that could act as a bridge, applicable in a broader range of indications, so that even in the case of progression, patients can continue treatment in the same hospital where their therapy began.

Some members of our team, as medical students, have personally witnessed the tragedy of patients with advanced HCC, who were excluded from transplantation. These experiences motivated us to search for a new therapeutic strategy that could change the outlook for such patients. We realized that synthetic biology provides tools that can be more selective, adaptable, and safer than traditional chemotherapy or systemic treatments. In particular, toehold switches – the synthetic biology mechanism we aim to use – allow for unprecedented specificity, opening the door to a targeted mRNA-based therapy that could precisely recognize and destroy cancer cells.

Our project is not only about science – it is also about people. HCC and the lack of effective therapies impact not only the health of patients, but also their families and the healthcare system as a whole. Our motivation goes beyond incremental improvements: we want to contribute to a paradigm shift in HCC therapy, so that every patient – regardless of whether they live near a transplant center or not – could access a treatment that gives them a real chance. To address such a complex challenge, we built a multidisciplinary team that includes students of medicine, biology, chemistry, bioinformatics, journalism, marketing, and graphic design. This diversity allows us not only to design an innovative project at the laboratory bench, but also to communicate it effectively to patients, clinicians, and the wider society.

Finally, our motivation is also educational. We aim to raise awareness in Poland about the potential of biotechnology and synthetic biology, and to inspire others to explore how these tools can be used to tackle pressing medical problems. Inspired by last year’s Polish team, PrymDetect, we decided to join the iGEM community and take part in iGEM 2025. Together, by combining computational modeling, cell biology, and synthetic biology, we aim to bring our project one step closer to real clinical application in the fight against HCC.

Cancer

Cancer is a disease humanity has faced since the dawn of time[1]. In the beginning, it was not very noticeable and did not cause a significant number of deaths, as infectious diseases were the main killers[2]. Medical progress allowed us to overcome most of these deadly illnesses and extend human life expectancy, leaving cancer as one of the last great opponents of medicine. Uncontrolled cell division – which is precisely what cancer is – has become the second most common cause of death in recent years. Although medicine has managed to find cures for some cancers, it remains powerless against many others[3]. One of these is hepatocellular carcinoma (HCC)[4]. [Figure 1.]

Figure 1
Figure 1. The Edwin Smith Papyrus from ancient Egypt, dating back to 1600 BCE, contains the first known description of cancer: “There seems to have been no treatment known to the physician, either for these tumors or for the same trouble appearing anywhere on the body.” (Refrence: Institute for the Study of Ancient Cultures, The University of Chicago, https://isac.uchicago.edu)

Hepatocellular carcinoma (HCC)

Hepatocellular carcinoma (HCC) [Figure 2.] is the most common primary malignant tumor of the liver and one of the deadliest cancers worldwide [5][Figure 3.][Figure 4.]. The most effective treatment today is surgical tumor resection or liver transplantation, with a 5-year survival rate of about 70%. Even when patients qualify according to the Milan criteria, the number of available donor organs is limited, and the tumor continues to grow, leading to patient disqualification from transplantation[6]. [Figure 5.] Unfortunately, despite advances in medicine and diagnostics, only about 30% of patients are diagnosed at an early stage, which qualifies them for surgical intervention[7]. The remaining 70% are diagnosed at an advanced stage, condemning them to palliative care[8]. In 2007, a new systemic drug, sorafenib, was introduced, which for the first time significantly extended survival from 7 months to an average of 12 months and became the gold standard in advanced HCC treatment[9]. Unfortunately, the 5-year survival rate remains very low, which is why there is an urgent need for a targeted therapeutic strategy to improve patient survival.

Figure 2
Figure 2. Liver with HCC (Source: https://www.lecturio.com/concepts/hepatocellular-carcinoma-hcc-and-liver-metastases/)
Figure 3
Figure 3. Every day, 2,300 people are diagnosed with HCC, of ​​which 1,600 people a day learn that it is in the advanced stage and that they have about a year and a half left to live[5,67]. 2,200 people die from HCC every day.
Figure 4
Figure 4. The main risk factors for the development of HCC include HCV, HBV infections and liver cirrhosis.
Figure 5
Figure 5. Distribution of lipid nanoparticles with encapsulated mRNA.

The problem of liposomes and the liver

In recent years the development of RNA vaccines marked a milestone in medicine, which gave hope for targeted anticancer therapies. Currently, the most effective delivery method is encapsulation of mRNA into lipid nanoparticles (LNP)[10]. Unfortunately, more than 70% of the total intravenously administered dose is captured by the liver, which significantly complicates treatment and poses a particular problem in the development of innovative CAR-T mRNA therapies[11],[12],[13]. [Figure 6.] Our project aims to turn this weakness into an advantage, since our therapeutic target is cells located in the liver. This enables a targeted approach not only at the molecular level but also at the level of the drug’s physiological distribution.

Figure 6
Figure 6. Pyroptosis mechanism.

Specificity of AFP

One of the main challenges in designing innovative targeted anticancer therapies is finding a specific protein – an antigen – that appears only on tumor cells and not on surrounding healthy cells. HCC, however, is a very specific tumor, as it produces alpha-fetoprotein (AFP)[14]. AFP is a fetal protein with transport functions similar to albumins, playing an important role in fetal development[15]. After birth, hepatocytes physiologically produce only very small amounts of AFP. An increase in AFP levels, however, is used in HCC diagnostics, since about 70% of patients are seropositive for AFP[16]. Our goal is to go one step further: to use AFP not only as a diagnostic marker but also as a therapeutic target.

How the iGEM community influenced our choice

Previous IGEM teams significantly influenced our project choice, and based on their results we were able to optimize our experiments. The Triggate program was the primary tool for designing our riboswitches. Thanks to its option to create toeholds for eukaryotic cells, we were able to design toeholds functional in human cells. Moreover, by integrating multiple NUPACK tools into a single platform, we were able to optimize riboswitch structures in a short time and analyze a greater number of variables.

The project of the UParis_BME team enabled us to design the first riboswitch using a full mammalian transcript as a trigger. Their research on miRNA allowed us to optimize our toeholds for human mRNA. The choice of effector protein encoded by our riboswitch was largely influenced by the Proteus team. Thanks to their studies, we understood that pyroptosis could be induced through our riboswitch and would provide the most effective therapeutic outcome.

Apoptosis vs. Necrosis → Pyroptosis

From the beginning of evolution, multicellular organisms had to deal with abnormal cells that disrupted homeostasis. The physiological solution to the problem of virus-infected or mutation-damaged cells is apoptosis – programmed cell death[17]. Apoptosis, whether induced endogenously by the cell or exogenously by the immune system, allows immunologically silent cell death. It does not cause inflammation and does not disrupt homeostasis[18]. Apoptosis is the most common form of cancer cell elimination induced by chemotherapeutics, since it does not overly activate the immune system[19].

In the case of HCC, however, apoptosis is problematic due to specific molecular mechanisms. HCC cells show, among others, overexpression of anti-apoptotic BCL-2 family proteins, activation of apoptosis inhibitors (XIAP), and activation of the NF-κB transcription factor. This makes apoptosis induction significantly more difficult than in many other cancers[20]. On the other hand, necrosis – uncontrolled cell death caused by severe damage – triggers chaotic and uncontrolled immune activation[21]. In tissues already devastated by cancer, this leads to significant destruction of healthy cells, negatively affecting patient survival[22]. An ideal solution combining programmed cell death with controlled immune activation is pyroptosis, described in 1992.

Pyroptosis, from Greek pyros (fire) and ptosis (fall/death), is based on caspase-1 activation. Caspase-1 activates gasdermin proteins that form pores in the cell membrane, leading to swelling and lysis. During pyroptosis, pro-inflammatory cytokines such as IL-1β and IL-18 are released into the local cellular environment. These stimulate the immune system in a controlled manner, allowing the clearance of all tumor cells without causing significant damage to healthy cells of the organ[23]. The first signal for pyroptosis comes from DAMPs/PAMPs, which activate the NLRP3 inflammasome. This inflammasome then activates caspase-1, which cleaves the final substrate of the pathway – gasdermin. After cleavage, the N-terminal fragment of gasdermin integrates into the cell membrane, forming pores, causing swelling and lysis[24]. [Figure 6.] Pyroptosis, thanks to its ability to bypass HCC resistance mechanisms against apoptosis and to break local immunosuppression, is an ideal solution for effectively eliminating HCC cells [Figure 7.].

To minimize variables that could disrupt pyroptosis, given the constant mutations in tumor cells, we chose the N-terminal gasdermin fragment itself as our effector to induce hepatocellular carcinoma cell death.

Figure 7
Figure 7. Mechanism of toehold induction of pyroptosis.

How to induce pyroptosis in HCC cells – Toeholds

One of the main challenges in creating innovative targeted therapies is drug specificity toward cancer cells. This is where toehold riboswitches, discovered in 2014 [25], come into play. Toeholds are synthetically engineered RNA sequences with a specific stem-loop structure. Depending on the presence of a “trigger” – a complementary mRNA molecule – the stem-loop changes conformation, enabling translation of the desired protein only when the trigger is present in the cell.

Over the past 10 years, toeholds have been used multiple times in research and by other IGEM teams, but the vast majority of work has focused on prokaryotic cells, where their function is easier to control and predict. Our team used available bioinformatics tools to design toeholds dedicated for eukaryotic cells.

The riboswitches created with the Triggate server use a unique architecture blocking the Kozak sequence after the hairpin, increasing specificity and solving issues with translation initiation inside the loop. Although the hairpin length differs depending on parameters, all designs achieved high stability as confirmed by NUPACK analysis. The target sequences are based on the full length of the AFP transcript, and were verified to not appear elsewhere in the human transcriptome. The overall design philosophy ensures specificity, selectivity, and a clear difference in activity between the OFF and ON states [Figure 8.].

Figure 8
Figure 8. Toehold generated using TrigGate software (https://l7gcea66wh.execute-api.us-east-1.amazonaws.com/). Toehold number 21 – visualization performed with the use of open source toolkit Mol*.

Innovation is also education

Our team includes biology teachers and tutors, so educating younger students and encouraging them to explore biology beyond textbooks was particularly important to us. The development of mass media and online learning inspired us to create an educational product tailored to our audience and accessible to everyone regardless of location.

To this end, we created an educational platform that guides students from basic biotechnology to advanced synthetic biology techniques at their own pace. However, it is impossible to fully understand biology without entering the laboratory. That’s why we partnered with the Biocenter for Science Education, which organizes the largest number of laboratory classes for students in Poland. Through this collaboration, the most engaged students who solved tasks on our platform had the opportunity to participate in laboratory classes in synthetic biology led by our team.

Impact on the iGEM community

Looking at the results of our experiment, we can proudly describe our project as a milestone for future teams and for synthetic biology as a whole. Thanks to our research, we are able to add to the arsenal of potential anticancer drugs an extremely precise tool – toeholds – and enable their use in treating other diseases.

Our team introduces new AFP-specific toeholds, which are the first toeholds for the full transcript in mammalian cells. This opens up an entirely new spectrum of possibilities for future teams to design novel synthetic diagnostic and therapeutic tools using mRNA specific to particular eukaryotic cells. We see great potential for applying this type of solution in preclinical research as well as in environmental studies, and we look forward with anticipation to seeing how future teams will make use of it.

Moreover, the method we employed for transfecting mammalian cells with a plasmid carrying the toehold significantly streamlines pilot studies and will be a major advantage for future teams when testing the functionality of their constructs.

The software we created can identify mutated transcripts in tumor cells of individual patients and use them as targets for personalized toehold riboswitches. It processes raw Nanopore sequencing data to detect transcripts that are differentially expressed across multiple tumor regions compared to controls, filtering out candidates with close paralogs to avoid false positives. Using these selected targets, the pipeline generates toehold switches, spligttin them into k-mers, and optimizing their structure. The final output includes the highest-scoring toeholds with detailed structural annotations, energy scores, and fully assembled sequences that include the Kozak sequence, AUG codon, and linkers. Additionally, the software can generate visualizations of predicted RNA secondary structures and ensemble defects. Thanks to a user-friendly interface, researchers can simply provide raw sequencing data and reporter sequences, while the pipeline automatically performs transcript selection, toehold construction, and structure optimization.

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