Our modelling, collaborations, and outreach & engagement included building a SimBiology model to execute our project, hosting the first ever MIT Mammalian Meetup, attending the New England iGEM Meetup held by Harvard iGEM, contributing to the Maastricht iGEM Journal, contributing to the URochester biomarker database. You can find more information on these interactions on their respective pages in the toolbar.
Integrated Human Practices:
Meeting with Dr. Rushina Shah
Throughout our project, with so much information that could be gathered with clicks in our simulation, we thought it was important to take a step back and assess how our work fit into the broader whole. Throughout our project, we consulted with various experts and stakeholders in the field of synthetic immunology, including perspectives from bioengineers, mechanical engineers, and ethicists.
During her PhD in mechanical engineering, Rushina worked on modelling control theory on immunological molecular systems within the body. We met Rushina very early on and learned about her work on cytokine interactions in rheumatoid arthritis, which influenced our early conceptions on how we could build a system around our project.
Meeting with Sebastian Palacios
Sebastian was integral in helping us learn the beginnings of modelling using differential equations in MATLAB. He gave us advice on what was reasonable to expect within a biological model, and was able to leverage his background in computational structures to help us make informed decisions on how to best collect and organize data.
Meeting with Dr. Noreen Wauford
We met with Noreen to gather her insight on strengths, specialities, and shortcomings of the PERSIST system. Noreen helped draw our attention to a flaw in our model--the potential crosstalk of Csy 4 and Cse 3, which we originally had as our third ERN at the crux of our AND gate. Along the way in evaluating a new candidate for this position, Noreen also helped us make an informed decision between the tradeoff of a nonspecific (Cas13d) versus inefficient (Cas6) ERN, of which we ultimately decided to test Cas6 for our system design. Most importantly, Noreen, as one of the pioneers of the PERSIST system, was an invaluable source of guidance for our circuit design and helped us think of our system as an off-by-default system. Before, we were considering looking at a split Zinc-finger system, which would have been more difficult to control and build. Her advice helped us rethink our circuit design. She also helped us find information on characterization of Hill fits for ERNs in PERSIST. Thanks, Noreen!
Meeting with Dr. Fabio Caliendo
Fabio was able to provide a stronger sense of real-world context for clinical therapies. He gave us the advice to modify our output protein from being a decoy receptor to being an antibody, as these are more specific, and have already been clinically developed for each of the cytokines tested; further, delocalized receptors would not likely retain their integrity when taken out of the transmembrane region. We had a concern of antibodies being a bit big to comfortably integrate with our circuits as is, so we worked with him to implement the design of single chain variable fragments (scFvs) instead, as these were a smaller hybrid of the different antibody chains found on a native antibody, as well as could provide less immunogenicity in a proposed treatment format. We further learned more details on potential crosstalk between nuclear transcription factors, and were informed about Dr. Yvonne Chen's paper on developing minimal promoters for technicalities.
On another note, Fabio inspired us to consider a variant of circuit architecture for future implementation when we can test our system in vitro: multimodal regulation. Currently, our system is operating regulation at the translational level: imposing control on mRNAs. However, Fabio showed us that systems with regulation at both the transcriptional and translational level can provide an amplifying effect in the ERN response--if this is something we observe as needing after in vitro experimentation, we will definitely consider such an architecture. Thanks Fabio!
NEGEM Feedback and Meeting with Dr. Françoise Baylis and Dr. Kenneth Oye
Now that we had a prototype of the system, what did we do with it?
We first shared our findings at a local New England iGEM meetup,which gave us the opportunity to share our findings with scientists at Harvard completely removed from our technology. It was a great experience because we not only got to hear excitement, but critically, concerns of our project. The postdoctorates and graduate students encouraged us to more carefully consider not simply our therapeutic functionality, but also its implementation and acceptance--at the biological and consumer scale. That last point about considering consumer acceptance is a cornerstone of iGEM, and one we wanted to consult about further before addressing its biological implementation.
So, we met with Dr. Francoise Baylis of Dalhousie University and Dr. Ken Oye of MIT, both renowned for their work in biological ethics. Coming out of hearing concerns from NEGEM, we stressed the limited capacity of our work--how it was only a potential target for hypothetical future research that could maybe happen experimentally. And at first, Dr. Baylis and Dr. Oye were taken aback. They asked us why we even undertook this project if we didn't believe it had potential to make positive change in the world, however small, not just existing as information. They taught us how to look at our project for its capacity, acknowledging its limitations, but not minimizing it as we started to do. With this advice, we stopped perceiving our project as just a model that works, and dove back into thinking of our project in terms of real world limitations as something that could make tangible change.
Meeting with Dr. Michael Birnbaum
As a final run through of our iterated prototype, Professor Michael Birnbaum of MIT met with us to discuss safety measures and provide feedback on overall circuit design. He brought up three main points to improve our system:
- Debating targeting the IL-1 receptor vs the IL1 cytokine itself based on recent studies
- Designing in a kill switch to turn off in order to prevent over IL1 suppression based on a case where a patient developed sepsis
- Ensuring that our AND gate functionality was maximized by making sure that our inputs did not occur simultaneously.
Refining the target of our system
Encouragingly, Professor Birnbaum noted that we were on the right track, and sent us several papers which were exploring IL-1 receptor blockers as a possible treatment for cytokine storms (Cauchois 2020), (Kaps 2020), (Khan 2020). He also discussed how these papers were looking at both receptor blockers and cytokine blockers, and asked us to consider (when we return to the lab) testing both of these options to evaluate effectiveness, and noted that switching these would not be the most difficult task. While we did not have the chance to implement this feedback this year, we found this helpful for considering how our system might evolve in the future.
Designing a kill switch
If our system was effective, this was a good thing. However, if we were to lose control over our system in our programmed range, this could be dangerous, as in with anakinra inducing sepsis in a patient (Kaps 2020). One of Dr. Birnbaum’s first questions to us was whether our system could “turn off”. While we had been thoroughly considering ideas like kill switches in our minds, we did not include a certain design computationally because we would have liked to characterize such an integral part of our system experimentally first. This moment was actually really great for us, because we had come full circle--from in our early days as a team brainstorming big idea features that we would like such a system to have, to a team working the nitty gritty details of a computational model, to a team thinking back about how we could implement those big picture, human features we had pushed off for later. Additionally, we relayed how we had designed our system to be dose dependent in whether it could mount an appropriate response or not in a COVID-19 cytokine storm. We additionally brought up some previous ideas we had had about integrating an extra input arm to the AND gate for SARS-Cov-2 spike protein as another layer of regulation.
Maximizing AND gate efficiency
Professor Birnbaum also asked us about ways to simplify our system, and an especially pertinent question was if IP-10 and MCP-3 were produced as part of the same pathway; if so, then the AND gate would not be contributing as much as we intended. Fortunately, based on our earlier research, we determined that this was unlikely and our AND gate would in fact, add value to our system. However, this was a great point that we will keep in mind when considering refining our system (for example, by adding a kill switch).
Cauchois, R., Koubi, M., Delarbre, D., Manet, C., Carvelli, J., Blasco, V. B., . . . Kaplanski, G. (2020). Early IL-1 receptor blockade in severe inflammatory respiratory failure complicating COVID-19. Proceedings of the National Academy of Sciences, 117(32), 18951-18953. doi:10.1073/pnas.2009017117
Kaps, L., Labenz, C., Grimm, D., Schwarting, A., Galle, P. R., & Schreiner, O. (2020). Treatment of cytokine storm syndrome with IL‐1 receptor antagonist anakinra in a patient with ARDS caused by COVID‐19 infection: A case report. Clinical Case Reports. doi:10.1002/ccr3.3307
Khan, N. A. (2020). Anakinra for severe forms of COVID-19. The Lancet Rheumatology, 2(10). doi:10.1016/s2665-9913(20)30273-3
This page was written by Sangita Vasikaran, Ethan Levy, and Rachel Shen