DESCRIPTION

Overview

Using synthetic biology to tackle real-world challenges demands a thoughtful and engaged approach especially when it concerns human therapeutics. Our team recognized the importance of delving deeply into how our project is in line with ethical and societal considerations, ensuring it is both responsible and beneficial. Throughout the year, this process pushed us to actively reflect on our progress and contemplate our project’s purpose, its potential impact, and how external factors might influence our work.

Closing the loop

From the beginning of our journey, the process of integrating feedback into our initial idea,was crucial but quite challenging. We often got caught up in the chaos of meetings and brainstorming sessions, not really taking in what we heard or knowing how to move forward. Driscoll's “What” Model helped us reflect on new knowledge and perspectives by breaking down our thinking into three simple key questions: “What?”, “So what?”, and “Now what?”. This allowed us to first identify new information we encountered, such as feedback from stakeholders or changes in scientific understanding that often got lost in a long conversation. Then, by asking “So what?”, we were able to analyze the importance of these insights and understand how they could impact our project’s direction and goals.Finally, the “Now what?” stage helped us decide on concrete steps to take, ensuring we could adapt our future plans based on what we learned. This structured reflection process made it easier for us to continuously improve our project and stay responsive to new developments.

Understanding the problem-Stakeholders
Wet Lab
Dry Lab
Outreach
Implementation

Understanding the problem-Stakeholders

Understanding the impact of Multiple Sclerosis (MS) in Greece and globally, requires recognizing its multifaceted nature. MS is a complex neurological condition that affects individuals in various, often unpredictable ways, impacting not just physical health but also quality of life, social dynamics, and economic stability. Addressing this challenge goes beyond healthcare; it involves a coordinated effort among diverse stakeholders each of whom plays a crucial role in advancing research, developing innovative therapies, and ensuring patient-centered care. Finding and engaging with most key stakeholders is essential to combine efforts toward the ultimate goal of developing a radical treatment and making it widely available.

We started brainstorming using the salience model, which helped us come up with a plan to connect with stakeholders, after identifying them. This model helps prioritize stakeholders in a project based on three factors:

  1. Power

    Can they influence the project?

  2. Legitimacy

    Are their needs and involvement appropriate or valid?

  3. Urgency

    Do they need immediate attention?

  • Dormant Stakeholders:
    • Attributes: Power only.
    • Description: These stakeholders have power but don’t show urgency or legitimacy. They aren’t currently involved but could become influential if their situation changes. For example, a government agency with the power to regulate but not actively doing so.
  • Discretionary Stakeholders:
    • Attributes: Legitimacy only.
    • Description: These stakeholders have a legitimate interest in the project but lack power and urgency. They are supportive but don't require immediate attention. An example could be a charity that supports a project’s mission but doesn't exert influence.
  • Demanding Stakeholders:
    • Attributes: Urgency only.
    • Description: They have urgent needs but lack power and legitimacy. Their demands might be seen as annoying since they don't have much influence. For instance, a minor user who complains loudly on social media without much impact.
  • Dominant Stakeholders:
    • Attributes: Power + Legitimacy.
    • Description: These stakeholders have both power and a legitimate stake in the project but aren't urgently demanding a ttention. They should be kept satisfied and well-informed. For example, a key investor who doesn't push for immediate changes.
  • Dangerous Stakeholders:
    • Attributes: Power + Urgency.
    • Description: They have both power and urgency but lack legitimacy. They could pose risks or threats to the project. For instance, a group that might sabotage a project because they oppose it strongly.
  • Dependent Stakeholders:
    • Attributes: Legitimacy + Urgency.
    • Description: These stakeholders have legitimate claims that are urgent but lack the power to enforce their demands. They rely on others to advocate for them. An example might be a local community affected by a project.
  • Definitive Stakeholders:
    • Attributes: Power + Legitimacy + Urgency.
    • Description: These are the most important stakeholders because they have power, legitimacy, and urgency. They need to be managed closely as their needs are critical. An example could be a regulatory body that can halt a project immediately.

By understanding these categories, we were able to better prioritize our time and efforts to effectively manage stakeholder relationships.

The salience model
Figure 1:Our Salience analysis results.

Wet Lab

Dr. Michail Aivaliotis, PI

What?: The previous iGEM team, with the help of our PI, helped build a diverse team of many different scientific perspectives at different stages of their educational journey. Our team included students of health sciences, engineering, and natural sciences, ranging from freshmen to seniors. After conducting literature research and brainstorming ideas, we decided on the first draft of NeuroMusketeer.

So what?: After the project selection we all were very passionate about Multiple Sclerosis, wanting to develop a single unit mechanism that combats the immune response with a cellular component which then releases a molecular one to treat the nerve damage. Therefore, we concluded that no matter what adjustments needed to be made, our ultimate goal would remain MS.

Now what?: We decided to start with feedback from academia and clinicians [notebook], on the cell and molecular pillars individually but at the same time, being open to any input because our project was at its very early stages .

Photo-of-our-team-meeting Photo-of-our-team-meeting
Photo-of-our-team-meeting
Marina Kleopatra Boziki, M.D, PhD
Assistant Professor, Multiple Sclerosis Centre, 2nd Department of Neurology, School of Medicine, Aristotle University of Thessaloniki

What?: We met with Ms. Boziki to present our project, NeuroMuSkeeter, focusing on our targets and how well they correlate with MS pathology and current therapeutic objectives. She informed us that targeting cytokines in the central nervous system (CNS) for MS has been proven ineffective on a clinical level and suggested myelin proteins instead. She approved of our choice of miRNAs and the integration of miR therapeutics in general but urged us to conduct a more comprehensive review, which she then validated.

So what?: Ms. Boziki's feedback highlighted critical considerations in our project design. Targeting the CNS directly, instead of peripheral cytokines, may be more beneficial for MS treatment. Additionally, the complexity of cellular interactions in the CNS suggests a need for more realistic model systems and careful evaluation of our current strategies, especially for the single unit plan.

Now what?: Moving forward, we reassessed our cell target choices and considered more suitable alternatives that directly impact the CNS, selecting MOG and MBP, and ultimately landing on the latter in our final design. We also revised our research strategy to ensure that the best possible miRNAs would be used and confirmed our initial findings. Finally, we started discussing separating the cellular and molecular mechanisms entirely thus creating a two-unit system.

Evangelia Yannaki, M.D, PhD
Director, Gene and Cell Therapy Center, Hematology-HCT Unit, "G. Papanikolaou” General Hospital of Thessaloniki

&
Anastasia Papadopoulou, PhD
Senior Researcher & Production manager, Gene and Cell Therapy Center, Hematology-HCT Unit, "G. Papanikolaou” General Hospital of Thessaloniki

What?: In our meetings with Ms. Yiannaki and Ms. Papadopoulou, we presented our NeuroMuSkeeter project. They suggested collecting T and B-cells from the blood instead of CSF for practicality and discussed re-administering these cells through the CSF for local effectiveness. They recommended using CAR-T cells while also highlighting the potential of gene editing technologies like CRISPR-Cas9 in T-cell immunotherapy, leading to the suggestion of FOXP3 epigenome regulation to induce Tregs from cytotoxic T cells.
Having liked our initiative, they also suggested hosting us for some of the necessary experiments and taking on a more active role, which we delightedly accepted.

So what?: These insights emphasized the need to reconsider our initial strategies, particularly in terms of cell receptor design. The suggestion to develop anti-CD19 CAR-Tcells and activate FOXP3 via gene editing, combines established methods with innovative techniques, potentially enhancing the therapeutic impact while minimizing logical gaps in our research.
Their expertise in cell therapies in both lab and clinical settings rendered their help and input an incredible asset.

Now what?: Moving forward, we will create a subgroup for the cell receptor part of the project and start collectively designing it and planning the experiments. Simultaneously, we will seek more feedback and help on the CRISPR/Cas9 component.

Photo-of-our-team-meeting
Photo-of-our-team-meeting
Nikoleta Psatha, PhD
Assistant Professor, School of Biology, Faculty of Sciences, Aristotle University of Thessaloniki

What?: After establishing our partnership, Mrs. Giannaki and Mrs. Papadopoulou, brought in Mrs. Psatha, to advise us on the CRISPR/Cas9 design and experiments. She also agreed to partner with us and host us in her lab, offering us the support of her whole team.

So what?: Adding another experienced molecular biologist to our scientific partners was an amazing development for the team. Not only was it a necessary helping hand for our wet lab activities, it also provided us with important bioinformatics knowledge which saved us a lot of time on the design.

Now what?: We will create a subgroup only for this part of the project and work closely with Mrs. Psatha and her research group.

Overall reflection and feedback integration

After several discussions and literature searches throughout the months of April and May, we concluded that a CAR design would be the best way to engineer our T cells to ensure efficacy and specificity. However, instead of a classic receptor targeting CD19, we decided to design a receptor that targets the autoantigens and autoreactive cells that induce MS (Chimer Auto Antibody Receptor, CAAR). The Tcells would also be epigenetically modified via CRISPR/Cas9 to become Tregs, thus ensuring a safer and more controlled alternative to the usually risky CAR-T cells therapies. Lastly, the miR hairpin structure would still be designed but its function would be separate from the cell.

Dry Lab

IT center, Aristotle University of Thessaloniki

What?:We contacted our university’s IT center that is responsible for all digital activity and spaces of students and staff alike. We wanted to inquire about the possibility of building a software interface that would be available and easy to use by the university community and guests. They briefed us on the front end development tools they use and explained to us the process of creating a website for our software tool. Furthermore, we asked about potential tools we may utilize to address the computational needs of our software machine learning model and they gave us information about potential, widely available ones such as Google Colab.

So what?: The IT center was willing to support and accommodate our request and gave us a comprehensive overview of what can and can’t be done with our current resources.

Now what?: With our new knowledge, the dry lab team can continue their work and training with more precise and calculated steps.

Photo-of-our-team-meeting
Photo-of-our-team-meeting
Andigoni Malousi, PhD
Bioinformatics Teaching Staff, School of Medicine, Aristotle University of Thessaloniki

What?: Ms. Malousi reviewed our idea from a theoretical standpoint and pointed out flaws to its structure (lack of repeated motifs in RBS structures) and output (number of compatibility strength categories). She also recommended various databases to gather model training data from and provided us with her research from a previous machine learning project of hers that is similar to our software tool.

So what?: Our idea’s initial structure and output could impede its practical application in biological systems and could produce misleading results for us and future users.

Now what?: We addressed the defects discovered by Ms. Malousi and redesigned an improved version of our software. Additionally, we delved into the recommended databases to gather the necessary training data and her previous research to improve the model architecture, ensuring our model is robust and accurate.

Ioanna Chouvarda, PhD
Associate Professor, Laboratory of Computing, Medical Informatics and Biomedical - Imaging Technologies, School of Medicine, Aristotle University of Thessaloniki

What?: Ms. Chouvarda gave us generally positive feedback and advised us on reviewing different machine learning models, techniques and architectures and their effectiveness, considering how they might be used together, and organizing the training data.

So what?: Getting approval from an experienced medical bioinformatician, meant that our software was headed in the right direction and we should continue improving upon the already established design.

Now what?: We thoroughly examined every idea Ms. Chouvarda provided and by implementing her suggestions, our model became more well-rounded and versatile. We successfully utilized her advice in designing our proposed model architecture for the Promoter Strength Prediction Model (PSP Model). Lastly, we effectively incorporated the predicted output from the PSP Model into the RBS Strength Prediction Software Tool, thus combining our two distinct software tools to enhance their performance.

Photo-of-our-team-meeting
Photo-of-our-team-meeting
Vasiliki Fadouloglou, PhD
Associate Professor, Department of Molecular Biology and Genetics, Democritus University of Thrace

What?: Ms. Fadouloglou provided crucial insights regarding the modeling aspect of our project. She recommended two programs for our work: Protein Data Bank (PDB), in order to research and analyze determined protein structures, we were advised to examine whether the proteins we intend to use, even if not fully defined, have known structures in separate regions, and PyMOL, a graphical tool to visualize these structures and assess their folding patterns. Regarding our concern about CD8a forming dimmers, she suggested using PyMOL to evaluate if the protein is amphipathic or not. We also inquired about predicting the functional percentage of our protein and the effectiveness of the signaling function. Ms. Fadouloglou clarified that such insights will primarily come from experimental results.

So what?: hese insights underscore the need of using computational tools for better structural understanding of our proteins before proceeding to experiments. Ms. Fadouloglou’s recommendations on PDB and PyMOL highlight the importance of visualizing protein structures in advance and ensuring that our proteins, even if partially defined, align with our design expectations.

Now what?: Moving forward, we used the PDB to identify the protein domains. After locating the domains, we retrieved their sequences, synthesized and ran them through AlphaFold v2 for structure prediction. We then used PyMOL, as outlined in the modeling process, to visualize the folding and assess the structural properties.

Outreach

Student’s Week AUTH , AUTH on Sundays ATP Conference, ReAcTiON Conference

What?:Our team decided to participate in university community activities, to touch base with the general public and fellow students, gather opinions and see whether or not our proposed solution is understood and supported by those it aims to benefit. However, we discovered that despite having researched the disease and SynBio thoroughly, we were not able to communicate our objectives in a universally understood manner.

So what?: Proper communication and public input is essential because it can guide the project to be more transparent, inclusive, and aligned with community values. When dealing with such a complex and sensitive issue, miscommunications or omissions, can leave major concerns unaddressed and create potentially dangerous discrepancies in our research.

Now what?: We concluded that in order to appropriately raise awareness and inform, we have to involve stakeholders with experience in public engagement that can help us present our work and goals.

Photo-of-our-team-meeting
Photo-of-our-team-meeting
Efi Theodoraki
General Directorate of Education and Lifelong Learning, Municipality of Thessaloniki

&
Vasileios Vasileiadis
Head of Department of Programmes and Lifelong Learning, Administration of Education,Municipality of Thessaloniki

What?: In our meeting with the Department of Education in our Municipality, they emphasized the need to educate the general public about Multiple Sclerosis (MS) in an engaging and respectful way. Their advice was to approach this as a local, humanitarian issue first and connect with MS communities in our region.

So what?: Understanding local perspectives ensures our outreach is relevant and effective. Partnering with MS communities and hosting events at accessible venues will boost awareness and support.

Now what?: With the help of our Municipality and Regional Directorates we will consult local MS patient groups.

Martha Sidiropoulou
MS patient, Member of "solidarity group for people with MS "

&
Anastasios G. Orologas, M.D
Chairman of Greek Multiple Sclerosis Society, Professor Emeritus of Neurology,School of Medicine, Aristotle University of Thessaloniki

&
Eva Ioannidou
Administration officer,Greek Multiple Sclerosis Society

&
Sofia Theodorou, Msc
Psychologist and Board Member, Greek Multiple Sclerosis Society

What?: Representatives of the Greek MS society and a local patient support group informed us about the social aspects of living with Multiple Sclerosis (MS), emphasizing the importance of supportive spaces and local awareness. They educated us on the current objectives of MS societies both nationally and globally, such as reducing stigma in smaller communities and advancing new treatments. Additionally, they advised us on how to establish contact with MS patients, and assisted in creating a questionnaire to understand their views, concerns, and knowledge about synthetic biology for MS treatment.

So what?: Understanding the social dynamics of MS and the objectives of MS societies provides critical context for our project, ensuring that it is sensitive and effective. Engaging directly with MS patients and gathering their input helps tailor our synthetic biology solutions to their needs and concerns. This increases the relevance and potential impact of our project, fostering better community support and acceptance.

Now what?: We will use the insights gained to refine our approach, utilizing the questionnaire to gather valuable feedback from MS patients and integrating their perspectives into our future implementation strategy.

Photo-of-our-team-meeting
Thessaloniki International Fair, Researcher’s Night

What?: Nearing the end of our iGEM journey, we decided to participate in distinctive events for our city and the greek scientific community respectively in order to implement what we learned about public engagement during the year. We improved our communication making it more understandable, concise and tailored to the person we are addressing.

So what?: It is essential to keep our outreach efforts up and use our newly found knowledge and skills to better inform the public and display our work.

Now what?: With the progress we have made and the experience we have gained, our goal is to keep introducing iGEM and NeuroMuSceteer at the best of our abilities to scientists, science enthusiasts and the broader community, locally and internationally.

Implementation

Grigoris K. Papagiannis
Dean (at the time), Faculty of Engineering, Aristotle University of Thessaloniki

What?: Our university’s engineering faculty is well known for its many student groups, a lot of which excel in international competitions and even go on to become successful businesses. The dean was thus able to give us tips on how to seek funding and present our team and work to attract interested parties.

So what?: Properly marketing ourselves and establishing a respected, professional presence is very important if we want to take our project to the next step. Long- standing, experienced university teams that have achieved that, pose a great role model that is also realistic to our circumstances. Therefore, reviewing their activities and conduct is a good way to draft our own game plan.

Now what?: After meeting the dean and getting his advice, we had a clearer idea of how to go about fundraising and outreach and resumed those activities more strategically. Meanwhile, inspired by our peers in the aforementioned engineering groups we started brainstorming business proposals.

Photo-of-our-team-meeting
Novartis Hellas
Chrysa Panagopoulou
Therapeutic Area Head Neuroscience Immunology

&
Eleonora-Iris Patista
Medical Advisor, Neuroscience

&
Dimitra Ignatiadi
Medical Operations Lead of Immunology and Neuroscience

What?: Representatives of the Greek MS society and a local patient support group informed us about the social aspects of living with Multiple Sclerosis (MS), emphasizing the importance of supportive spaces and local awareness. They educated us on the current objectives of MS societies both nationally and globally, such as reducing stigma in smaller communities and advancing new treatments. Additionally, they advised us on how to establish contact with MS patients, and assisted in creating a questionnaire to understand their views, concerns, and knowledge about synthetic biology for MS treatment.

So what?: Understanding the social dynamics of MS and the objectives of MS societies provides critical context for our project, ensuring that it is sensitive and effective. Engaging directly with MS patients and gathering their input helps tailor our synthetic biology solutions to their needs and concerns. This increases the relevance and potential impact of our project, fostering better community support and acceptance.

Now what?: We will use the insights gained to refine our approach, utilizing the questionnaire to gather valuable feedback from MS patients and integrating their perspectives into our future implementation strategy.

Eleutheria Papanikolaou
Technology Transfer Office, Research Committee, AUTh

&
Byron Chrysovergis
Technology Transfer Office, Research Committee, AUTh

What?: The technology transfer office is the link between academic research and industrial application. It promotes the conversion of research results into patents and licenses while supporting spin-offs and enhancing project contracts for public and private organizations. They looked at our business plan draft and helped us finesse it, filling in important information about intellectual property guidelines, paperwork and due diligence processes.

So what?: The implementation of an academic research project in the real world requires a lot of administrational and technical support. The department of our university in charge of this exact procedure, are the only ones who can thoroughly inform us on our commercialisation options, the necessary novelty assessments and what information is proprietary.

Now what?: After our meeting we had a direct scalability objective as well as the right tools to review our IP and put our patent portfolio together.

Photo-of-our-team-meeting