Novel logic gates in mammalian cells based on genetically incorporated unnatural amino acids

Novel logic gates in mammalian cells can expand the scope of synthetic biology, benefiting the society. Synthetic biology focus on building artificial biological systems for research, engineering and medical applications. Logic gates are often the foundation of these applications. For example, synthetic biologists have used logic gates to construct cell-based sensors for detecting environmental pollutants, toxic chemicals, pathogens, cancer cells, etc. All of which have direct benefit to the society. In addition, logic gates can also be used to develop novel therapeutics. In fact, medical application represents one of the most exciting areas for synthetic biology.

The research we wish to carry out here is to engineer novel logic gates in mammalian cells. These logic gates will process the input signals to produce an output signal in mammalian cells. These signals are binary (e.g. yes or no). In our design, the input signals will be the presence or absence of small molecules that otherwise have no effects to the cells. This characteristic is important so that the molecules can be used solely to control the logic gate output without interfering any cellular processes. In our design, the output signal will be functional or not of a protein. We wish to engineer logic gates that process the input signals rapidly and adjust its output signal accordingly upon change of the input signals, like a simple small computer. These features mean that the proposed logic gates will be particularly useful for applications where reversible fine regulation of a protein function or a cellular event is required but difficult to achieve by existing technologies.

We will use amino acids that do not exist in nature as the small molecules for the input signals. Nature uses 20 amino acids as the building blocks to construct proteins in our body. Here, we intend to use unnatural amino acids that are not toxic and pose no observable effects to cells. More importantly, using a special technique of our expertise, these unnatural amino acids can be inserted into specific position of a protein at our wish. We have used this technique to control protein function and gene editing by the presence or absence of an unnatural amino acid. However, this technique has never been applied for logic gate engineering. The proposed logic gates are thus novel and will also be complementary to those currently available. It is therefore possible to assemble complex genetic circuits using different logic gates.

In this project, we will engineer the basic logic gates that perform different logical operations. These basic logic gates can be combined to perform sophisticated tasks and are the basis of more complex logic gates. We will construct them in mammalian cells and characterise their performance using different analytical techniques. We will also implement a logic gate to control the function of engineered immune cells. Immune cells are part of our body's defence system. They can be engineered to combat non-infectious diseases, like cancer. Such cell-based therapies are of great promise and are provided by the NHS for children and young people with B cell acute lymphoblastic leukaemia. In some case, the cell-based therapies have even cured people where all other treatments have failed. However, they could also cause adverse side effects and even patient death. Although biological investigations and medical applications are outside the scope of present proposal, the logic gates to be developed here could improve the safety of current cell-based therapies, addressing a key concern of doctors and patients.

Overall, we propose to engineer novel logic gates in mammalian cells. These logic gates will respond to non-toxic unnatural amino acid and can be used for reversible fine regulation of a protein function or a cellular property. The proposed logic gates have potential in different biomedical application and will likely have direct benefit to the society.

The proposed research will have significant impacts on (i) synthetic biology and (ii) medical science as described in the Academic Beneficiaries.

According to Transparency Market Research, the global cancer immunotherapy market is going to grow from £30 billion in 2015 to £100 billion by 2025. CART-T therapy, a cell-based immunotherapy recognised as the next generation cancer therapy, is considered to make significant contribution to the market value in the upcoming years. Although the future of CAR-T cell therapy holds incredible promise to revolutionise our approach to treat different cancers, side effects from the CART-T therapy are not uncommon. In the worst case, the side effects can cause significant toxicities and lead to sudden patient death. Such an outcome is undesirable for not only the patient but also the patient's close families and friends, as well as the hospital and the pharmaceutical company which could be liable for significant compensation. Thus, new approach to better manage toxicities, such as the one proposed in this project, is needed and will benefit all parties in society.

A growing number of logic gates in synthetic biology have been engineered and characterised. Although much of this research and development is still very fundamental, examples of socially useful applications have emerged. For example, synthetic biologists have used logic gate to control production of fine chemicals in cells. Logic gate has also been used to construct cell-based sensor for detecting environmental pollutant, toxic chemicals, pathogenic organisms and even cancer cells. Some of the cell-based sensors could even be turned into therapeutics to fight diseases in humans. Though major successes in synthetic biology-based applications are yet to emerge, the payoffs are predicted to be significant. There are currently unmet needs to engineer logic gates with high processing speed, as well as different combinations and functionalities for biotechnological applications. This project will directly contribute to the needs in the field, providing a foundation for precision engineering to construct more complex biological systems in future.

Communication to industry will partly be through the same mechanisms as described in Academic Beneficiaries. In addition, the PI will present in conferences, such as CAR-TCR Summit, attended by mostly industrial leaders. In fact, the PI already had conversation with Celyad and Leucid Bio, biopharmaceutical companies focusing on the development of CAR-T therapies for the treatment of different cancers. Whenever suitable, we will seek exposure on popular press. The PI was a British Science Association Media Fellow in 2018. He had worked with the correspondence team in BBC Wales, providing contents for TV, radio and online news programmes, and is very familiar with the media culture.

We will periodically discuss our progress and findings with the Research and Innovation Services department at the Cardiff University. The Research and Innovation Services department will assess any outcomes that should be protected as intellectual property and also help to initiate contact with potential industrial partners. The department is well equipped to protect intellectual property, set up license arrangements and handle all aspects of commercial exploitation in this project. We will therefore be at a prime position to forge collaborations with industrial stakeholders and push our technology in a more translational direction.

Communication of the results to government will be through PI's participation in annual Life Sciences Research Network Wales congress and the annual Science and the Assembly event with Welsh Government. These events involve government representatives and experts from Public Health Wales, providing a direct route to communicate with independent policy bodies and other professional groups.