Team:Fudan/Human Practices

2018 iGEM Team:Fudan - Human practices

Human practices

Our ENABLE team as one seeks to advocate the bright future brought by synthetic biology, to attract and to motivate people to join us to work together.

Human practices

Our ENABLE team as one seeks to advocate the bright future brought by synthetic biology, to attract and to motivate people to join us to work together.

Human as social beings always has diverse motives and intentions. Our ENABLE team as one seeks to create tools that could readily be used by others. Our ENABLE team as one seeks to advocate the bright future brought by synthetic biology, to attract and to motivate people to join us and work together. Our ENABLE team as one seeks to transform the world by our own hardworking.

Interview doctors with different backgrounds to understand their common needs

Getting feedback from researchers of different fields helps us to improve our project continuously.


The four industries most interested in synthetic biology, and their share of the total number of surveys who is interested in synthetic biology


Among the four industries most interested in synthetic biology, the proportion of those who are familiar with synthetic biology accounts for the total 585 people surveyed.

We believe that to make our project widely applicated in the future, the first thing we need to do is to engage with our potential users fully. In this process, we learned about their views and expectations, both on our project and our future applications, thus providing timely feedback to our project design.

Immediately after we had come up with the overall draft of our project, we contacted some renowned people in clinical medicine, which we believe could benefit most from our project. We interviewed and discussed with Dr. Xinjun Yu from the Institute of Immunology, Dr. Lixin Yang from the Department of Thoracic Surgery at Changhai Hospital, Dr. Xiaoying Bi from Department of Neurology of Changhai Hospital and Dr. Pengfei Luo from the Burn Research Center. Their feedback could help us a lot in the optimization of our project.

Dr. Yu from the Institute of Immunology

    As an established immune cell researcher, his comments were mainly on comparison between our project and CAR-T. From the perspective of his research, he gave us several enlightening suggestions, summarized as follows:
  • Compared to CAR-T, our project could make immune cells perform more complex tasks, or even solid tumor treatment, where CAR-T has limited effect
  • Regarding application, we can try to achieve an assorted ‘army’ of immune cells for combined cancer treatment
  • The early design of our logic circuits was not unified. This might cause inconveniences to users and limit its usage
  • We have to form a systematic debugging process, to reduce the experimental difficulty and improve efficiency in the lab

Dr. Bi from Department of Neurology of Changhai Hospital

Director Bi, as an expert in neurology, has a great interest in synthetic biology. She fully affirmed the potential of our project, but also expressed her doubts. She pointed out that the intensity of many cell membrane surface ligand in the brain might not be as strong as we had expected, especially in the early stages of disease development, so the input signal is often very weak. However, the early stage of neurological diseases is the most crucial time for successful treatment with better recovery potential. Therefore, if the clinical neurological applications of our project should work ideally, it requires us to adjust our system for higher sensitivity. Intrigued and excited by our project, she delightfully invited us to have another round of discussion after iGEM.

Dr. Yang from the Department of Thoracic Surgery

    Director Yang has high hopes for our project’s application in precise cancer therapy. As an experienced surgeon and a clinical oncologist, he expressed his expectations and anxiety for new cancer treatments. His main concern was about the effectiveness and safety of our tools in cancer treatment, as summarized below:
  • The more comprehensive function immune cells achieve, the larger and more complex the logic system has to be. The construction and delivery of such a system will be a big challenge.
  • Simplify and unify our design as much as possible to enhance robustness and to avoid unforeseen problems.
  • Enhance our target precision to prevent unnecessary damage to healthy tissues

Dr. Pengfei Luo from the Burn Research Center

Dr. Luo mainly engages in translational medical research on burnt skin regeneration. He proposed that one application of our project could try to achieve precisely controlled skin regeneration so that the repair activities of the engineered cells can be activated or terminated under certain situations. After this fruitful interview, we expressed our willingness to the relevant application design after we completed the project improvement, and our proposal was welcomed by him. At the same time, he put forward the following suggestions to us:
In the transmembrane signal transduction process, the loss of signal may cause the activation signal to be too weak to activate the expression of the downstream target.
In our early project design, some logic gates contain too many layers of regulation and expression. Therefore, it may face signal interference problems.

The interviews with doctors and researchers in other fields continue. We have already received a lot of feedbacks (click to view the transcripts), from experts in different areas and represent the broad expectations of our adopters.

Logic gate redesign & SynNotch optimization

By talking to different people, outside fo team (actually outside of our university), we found many shortcomings in the logic gate genetic circuits we originally proposed. After literature searching, extensive discussions and debates, we have our ENABLE toolbox with 3-layer design.

We summarize the improvements we have made as follows:

Previous After improvement
Cannot control the expression of endogenous genes The third layer, Combiner, can be applied to control endogenous gene expression
The genetic circuit of some logic gates are very complex more than three layers We unified our design to have Receptor-Amplifier-Combiner 3 layers
The SynNotch intracellular domain of each logic gate varies All the logic gates share the same SynNotch intracellular domain for the orthogonal application

For more details, please visit: Improve, Measurement, Optimization, Results pages.

Make our project accessible

The purpose of any scientific research should be to understand the world better, solve problems, and provide unlimited possibilities. Our project aims to understand the contact-dependent interactions between mammalian cells, as well as to provide a toolbox of eukaryotic transmembrane logic gates. Thus, we explored the possibilities of our project with experts in other research fields together.

Functional repair of damaged brain tissue after stroke

During our first joint meeting with a team of neurology clinicians in Changhai Hospital, we learned some of the main problems in neurobiology research and clinical treatment. On our second joint meeting with them, we discussed in detail the application prospects of our project.

Stroke, as a high-risk cerebrovascular disease, has the characteristics of rapid onset, high mortality and poor prognosis. More importantly, the incidence of stroke has gradually increased in recent years and is becoming one of the leading causes of death and paralysis.

Unfortunately, current treatments can only palliate damage to brain tissue through early hemostasis/vascular dredges. Early damage prevention and functional repair of brain tissue have not been realized, which is one of the biggest problems encountered in neurology.

    Attempts have been made to repair brain tissue damage using stem cells. However, this approach has the following disadvantages:
  • Stem cells that are not precisely controlled, therefore have a risk of tumor formation in the body
  • The effect of stem cell treatment is questionable, and the mechanism of its effect in vivo is not clear
  • At present, stem cells are mainly transported to the lesion through stereotactic annotation, which cannot treat deep brain damage
  • The inner/periphery of the brain tissue after necrosis lacks the microenvironment that stimulates stem cells to differentiate into neurons. Most necrotic brain tissue is filled with astrocytes and loses its function.

We proposed to engineer neural stem cells to help the recovery after stroke.

    Cross the blood-brain barrier, with specific tissue targeting.
  • AQP4 (Aquaporin-4) is exclusively expressed on astrocytes (Saadoun S, et al.), so neural stem cells containing an anti-AQP4 extra-segment can specifically target to neural tissue
  • Engineered neural stem cell cells will not be activated when there is no cell necrosis in the nerve tissue
  • ALCAM (activated leukocyte cell adhesion molecule) guides edited neural stem cells to specifically bind to the blood-brain barrier and assist neural stem cells to cross the blood-brain barrier (Samaha H, et al.)
    Transmembrane AND gate enables suitable localization of neural stem cell differentiation
  • Brain tissue damage after a stroke, S100B is highly expressed in brain tissue (Czeisler BM, et al.)
  • The conjugated antibody binds to P57NTR (75kD-neurotrophin receptor) and the anti-s100B-SynNotch after being injected into the human body. On the one hand, it can repress P57NTR’s function of mediating apoptosis (Roybal KT, et al., Sergeeva SP, et al.) , on the other hand, it activates AND GATE in neural stem cells via anti-S100B--SynNotch
  • AND GATE then activates the expression of downstream neurotrophic factors in the neural stem cell, to promote neural stem cells to differentiate into neurons (Canals I, et al., Anderson MA, et al.).

    This ENABLE solution enables sensitive identification of brain damage and the prevention of further injury (such as cerebral hemorrhage and apoptosis activation) in a timely manner while creating a microenvironment suitable for neuronal differentiation. After completing the corresponding task, the circuit will be off, and the system will not run out of control.
    It has the following advantages:
  • Accurate positioning of engineered cells, and work status reports for visualization of treatment stage
  • Provide a suitable microenvironment for neuronal repair
  • The repair process is controllable and can be terminated at a given time
  • It could be injected into the brain in advance for patients with high risk of brain diseases, to protect and repair brain tissue immediately after the damage, to act as vaccines.

Smart skin regeneration

In our discussions with Dr. Luo, we learned that the current tissue differentiation is mostly done in vitro, which leads to morphological mismatch and possible risk of immune rejection. Especially in skin replantation, in vitro induction means that patients need to endure long waits and insufficient efficiency of skin grafting. Also, the natural repair relies heavily on the previously infected tissue, which means that it is currently impossible to achieve complete sterility of the patient's skin surface. There is an urgent need in the clinic for an alternative method for skin replantation.

From Dr. Luo’s unpublished research results, we learned that Smad3 is an essential signaling factor in skin regeneration. Its expression determines the differentiation fate of cells in different parts. We propose to build a Smad3 NIMPLY gate for mesenchymal cells. Engineered cells will achieve different differentiation stages at different sites, to make smart skin regeneration.

  • Under normal conditions (TLR4 is not expressed in skin cells), the ENABLE cells remains silent.

  • When the skin is damaged, TLR4 is expressed on the cell surface (Takeuchi O, et al.). When only TLR4 is present, our NIMPLY gate recognizes it and activates the downstream Smad3 expression. When Smad3 is highly expressed, the edited mesenchymal cells differentiate into fibroblasts, which will repair the dermis.

  • MCH Class II expresses on melanocytes and Langerhans cells, but not on fibroblasts (Plonka PM, et al.). When the repair proceeds to the epidermal layer, the edited cells recognize both TLR4 and MCH Class II, NIMPLY gate will down-regulate the Smad3 expression. Mesenchymal cells that recognize MCH Class II also express MCH Class II, which inhibits the expression of Smad3 once cells have already differentiated.

The smart skin repair solution we designed has the following advantages:
1. Respond quickly after skin burns, and can, to some extent, reduce secondary damage to the skin;
2. Enable different responses in different layers of skin, promote the repair of the epidermis, avoid the proliferation of the dermis and prevent the appearance of scar tissue;
3. Simulate skin repair within the damaged area as much as possible. This avoids mismatch caused by skin replantation;
4. Minimize the possibility of infection during surgical operations

Some of these applications still have a long to be realized, but they are feasible and could help these doctors solve the most urgent problems in their fields. Most importantly, we have demonstrated a new way to make the research results of an iGEM project understood by others - by pseudo-using it,

We believe that, to a certain extent, this kind of interaction between scientists and potential customers not only can be adopted by other iGEM teams but also help fundamental scientific discoveries to transform to clinical applications efficiently.

Impressed by the openness and willingness of the public

We organized a multi-disciplinary public debate, to gain ethics and safety insights from the public.

Before the real-world implementation of our project, ethics and safety should not be neglected. The public is undoubted pays close attention to these two aspects. More importantly, any valuable innovation ultimately needs to be transformed for public use or benefit. It was why we chose to hold a multi-disciplinary debate to gather ideas from more people. For more details, please visit: Public Engagement.

    Through debates, we found that the public focus on the safety could be improved by project design：
  • Prove orthogonality between promoters
  • Improve the sensitivity of SynNotch and reduce background-activation

We have ensured both. For more details, please visit: Improve, Optimization, Results pages.

Funded by the public, and to benefit the public

In September, we conducted an Internet survey and collected results from over 580 people with different backgrounds. We found that these people are very interested in synthetic biology, but have difficulty to understand the text we provided. We hosted the Bio-Art display with Legos (please follow the link to check more details). With hands-on logic gates building experience, the Display publicized the modularity of the project, and we learned how to convey synthetic biology to the general public effectively.

We have interviewed doctors with different backgrounds to understand their common needs, and brainstormed concrete applications of our project, such as facilitating brain repair after stroke and promoting skin regeneration. We want other scientists could readily use our project; thus we consult with many experts, including structure biologists who work on membrane receptors. By interacting with different people, we have been continually inspired through communications and dialogs.

Understanding is the base of trust and support. We did a quick experiment by running a-week long crowdfunding campaign (10/4 - 10/9). We described our project and potential applications, with links to our wiki pages. To our surprise, over 200 Internet backers in 6 days, although some of them are our friends and relatives. Agree well with our public debate; the public is very open and willing to try. A better living and a healthy body are all they want. We must always remember why we initiated the research and why we work hard in the lab - for the good of the people, including ourselves.

References

  1. Anderson MA, O'Shea TM, Burda JE, ..., Courtine G, Sofroniew MV. Required growth facilitators propel axon regeneration across complete spinal cord injury. Nature, 2018 Sep;561(7723):396-400 PMID: 30158698; DOI: 10.1038/s41586-018-0467-6
  2. Canals I, Ginisty A, Quist E, ..., Bengzon J, Ahlenius H. Rapid and efficient induction of functional astrocytes from human pluripotent stem cells. Nat Methods, 2018 Sep;15(9):693-696 PMID: 30127505; DOI: 10.1038/s41592-018-0103-2
  3. Czeisler BM, Janigro D. Reading and writing the blood-brain barrier: relevance to therapeutics. Recent Pat CNS Drug Discov, 2006 Jun;1(2):157-73 PMID: 18221201
  4. Plonka PM, Passeron T, Brenner M, ..., Hearing VJ, Schallreuter KU. What are melanocytes really doing all day long...? Exp Dermatol, 2009 Sep;18(9):799-819 PMID: 19659579; DOI: 10.1111/j.1600-0625.2009.00912.x
  5. Roybal KT, Rupp LJ, Morsut L, ..., Park JS, Lim WA. Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits. Cell, 2016 Feb;164(4):770-9 PMID: 26830879; DOI: 10.1016/j.cell.2016.01.011
  6. Saadoun S, Papadopoulos MC. Aquaporin-4 in brain and spinal cord oedema. Neuroscience, 2010 Jul;168(4):1036-46 PMID: 19682555; DOI: 10.1016/j.neuroscience.2009.08.019
  7. Samaha H, Pignata A, Fousek K, ..., El-Naggar S, Ahmed N. A homing system targets therapeutic T cells to brain cancer. Nature, 2018 Sep;561(7723):331-337 PMID: 30185905; DOI: 10.1038/s41586-018-0499-y
  8. Sergeeva SP, Litvickiy PF, Gultyaev MM, Savin AA, Breslavich ID. To the Fas-induced neurons apoptosis mechanisms in stroke pathogenesis. Patol Fiziol Eksp Ter, 2013 Jul-Sep;(3):15-8 PMID: 24340612
  9. Takeuchi O, Akira S. Toll-like receptors; their physiological role and signal transduction system. Int Immunopharmacol, 2001 Apr;1(4):625-35 PMID: 11357875
2018 team Fudan abstract

Abstract

Contact-dependent signaling is critical for multicellular biological events, yet customizing contact-dependent signal transduction between cells remains challenging. Here we have developed the ENABLE toolbox, a complete set of transmembrane binary logic gates. Each gate consists of 3 layers: Receptor, Amplifier, and Combiner. We first optimized synthetic Notch receptors to enable cells to respond to different signals across the membrane reliably. These signals, individually amplified intracellularly by transcription, are further combined for computing. Our engineered zinc finger-based transcription factors perform binary computation and output designed products. In summary, we have combined spatially different signals in mammalian cells, and revealed new potentials for biological oscillators, tissue engineering, cancer treatments, bio-computing, etc. ENABLE is a toolbox for constructing contact-dependent signaling networks in mammals. The 3-layer design principle underlying ENABLE empowers any future development of transmembrane logic circuits, thus contributes a foundational advance to Synthetic Biology.