Difference between revisions of "Team:MIT/Poster"
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<div class="title"> Medal Requirements </div> | <div class="title"> Medal Requirements </div> | ||
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<p>Developed a computational compartmentalized cytokine network that can be modified for a variety of cytokine-targeting treatments for inflammatory diseases with ten cytokines. </p> | <p>Developed a computational compartmentalized cytokine network that can be modified for a variety of cytokine-targeting treatments for inflammatory diseases with ten cytokines. </p> | ||
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<p>Hosted the international MMM (MIT Mammalian Meetup) with 18 registered teams.</p> | <p>Hosted the international MMM (MIT Mammalian Meetup) with 18 registered teams.</p> | ||
<p>Successfully simulated the cytokine storm system with ten cytokines. </p> | <p>Successfully simulated the cytokine storm system with ten cytokines. </p> | ||
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| − | < | + | <h3> Gold: </h3> |
<p> Engineering success integrated by reiterating circuit design through discussions with Weiss Lab. </p> | <p> Engineering success integrated by reiterating circuit design through discussions with Weiss Lab. </p> | ||
<p> Integrated human practices through discussions of implementation with the biological engineering community including bioethicists. </p> | <p> Integrated human practices through discussions of implementation with the biological engineering community including bioethicists. </p> | ||
Revision as of 17:32, 5 November 2020
MIT: Mammalian Circuitry for Treatment of COVID-19 Cytokine Storms
To summarize the full circuit depicted in Figure 3, we will sense the cytokines IP-10 and MCP-3 which correspond to the transcription factors ELK-1 and NF-κB respectively. CasE is expressed upon activation by ELK-1, and Csy4 is expressed upon activation by NF-kB. Using the CMV promoter, Cas6 is constitutively expressed but when either or both CasE and Csy4 are present, the mRNA for Cas6 will be cut upstream of the gene sequence, so Cas6 expression will decrease. For our output fusion protein, we have another plasmid with the constitutive promoter CMV, and likewise there is an ERN cut site upstream of the gene sequence. Thus, when both IP-10 and MCP-3 are present, scFv will be produced, inhibiting our cytokine of interest.
We developed an AND gate with five PERSIST-based plasmids. IP-10 and MCP-3 signal downstream phosphorylation pathways to activate nuclear transcription factors (nTFs) ELK-1 and NF-κB, respectively. Two minimal constitutive promoters (here, CytoMegaloVirus, CMV) are then induced by nTF-responsive element binding, upstream of ERN genes. In choosing our ERN components for translation, we evaluated the sensitivity with the relative abundance of the biomarker (nTFs ELK-1 and NF-κB) in plasma. In CCS, MCP-3 has been observed about 103 fold lower than IP-10; Csy4, an experimentally stronger “OFF” ERN, was thus placed downstream of the NF-KB-responsive promoter, while CasE, a slightly weaker ERN, was used for the IP-10 sginaling pathway. Cas6, the weakest ERN of the Cas family evaluated, when produced by either of two CMV-constitutively-expressed transcripts, degrades the scFv mRNA transcripts and causes decreased translation of anti-inflammatory soluble scFv with a slight buffer to allow for IP-10 and MCP-3 elevation to be reached. To further enhance the system dynamics by decreasing the time required to reach steady state, a PEST degradation tag can be fused to the Cas6 sequence. Our AND gate is thus engineered to produce a dose-responsive output when there is adequate IP-10 and MCP-3 to result in the degradation of Cas6 transcripts.
Now that we had designed our circuit, it was time to see how our components worked together. To do this, we simulated the behavior of our system in MATLAB‘s SimBiology compartmentally and mechanistically through differential equations. By compartmental, we mean that each reaction or event takes place in a defined location in respect to our engineered cell. In our model figure here, the intracellular space is orange, while the extracellular space is blue. By mechanistic, we mean that each step of a process is represented through interactions of species in different combinations of reactions, which are depicted as lines, as you can see from the legend on the left. Let’s take a closer look at the orange intracellular compartment.
Within our engineered cell, you can see the full circuit that, here in SimBiology representation. To put together the components, we thus constructed a system of differential equations that could model the interrelationships between species. A typical differential equation for a species looks like the one in blue text on the right: here, we can relate the rate of change of a species by adding its basal transcription, beta, subtracting its degradation, delta, and if the species participates in a binding event, a Hill cooperativity term. In the end, by providing literature values and relevant initial concentrations across timesteps, we can start to probe trends within the reactions we care about. If you look at the bottom of our AND gate, you can see this sort of antibody shaped representation of a translated ScFv output.
We can see the ScFv being exported out of our engineered cell into the extracellular space. This means we can assess its relationship to systemic serum circulating in the body. We were able to construct a cytokine interaction map in our model based upon plasma cytokine levels reported by Yiu et al. in patients undergoing inflammatory cytokine storms. We then could take our output ScFv and have it target each specific player in the cytokine storm, and from there, analyze its effects on the cytokine storm as a whole--using this information to tailor the future design of our system.
Bronze:
Developed a computational compartmentalized cytokine network that can be modified for a variety of cytokine-targeting treatments for inflammatory diseases with ten cytokines.
Characterized 15 parts associated with the PERSIST system from the Weiss Lab (Bba_K3621000-15) which can be used by future teams in mammalian circuits.
Silver:
Hosted the international MMM (MIT Mammalian Meetup) with 18 registered teams.
Successfully simulated the cytokine storm system with ten cytokines.
Contributed one review article and one original research article to the Maastricht iGEM Journal.
Contributed to the URochester Biomarker Database for characterizing cytokine storms.
Gold:
Engineering success integrated by reiterating circuit design through discussions with Weiss Lab.
Integrated human practices through discussions of implementation with the biological engineering community including bioethicists.
Successfully modeled the cytokine storm and tested different cytokine targets to identify two therapeutic targets.
Our modelling results can be seen in the graph above. We found two distinct features in our system's cytokine modulation: differences in the peak IP-10 observed and in the steady state value of IP-10.
First, we looked for the lowest peak IP-10 values, since this would indicate which therapeutic target would diminish the cytokine storm the most during the peak of the cytokine storm. As seen in the figure above, the lowest peaks corresponded to IFN-γ and IL-1, respectively; thus we found IFN-γ and IL-1 to be highly effective therapeutic targets for our scFv.
Another state we looked into was the steady-state levels of IP-10. These are significant since our modulatory system should bring the resting cytokine levels to a minimum. As seen in the figure, IL-1 and IFN-γ’s steady state had the lowest average values. Thus, we once again identified IFN-γ and IL-1 as strong candidates for our scFv target since over the long term, they would most effectively reduce the cytokine storm.
We found IFN-γ to most directly inhibit the cytokine storm. However, IFN-γ is also a master regulator of the immune response, and targeting this could lead to a significant drop in patient immune strength. (Kaps et. al.). Although IL-1 is also a key player in the immune response, other studies have found clinical efficacy in IL-1 inhibition (Cavalli et al., 2020). Thus, since IL-1 had the lowest steady-state IP-10 level, the second lowest peak cytokine level, and previous literature indicating its efficacy as a cytokine storm target, we found it to be the most effective target for our scFv in modulating the cytokine storm.
Acharya, D., Liu, G., & Gack, M. U. (2020). Dysregulation of type I interferon responses in COVID-19. Nat Rev Immunol 20, 397–398 doi:10.1038/s41577-020-0346-x
Cavalli, G., Luca G. D., Campochiaro C., Della-Torre E., Ripa M., Canetti M., … Dagna L. (2020). Interleukin-1 blockade with high-dose anakinra in patients with COVID-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study. The Lancet Rheumatology, 2(6), 325-331. doi:10.1016/S2665-9913(20)30127-2
Kaps, L, Labenz, C, Grimm, D, Schwarting, A, Galle, PR, Schreiner, O. Treatment of cytokine storm syndrome with IL‐1 receptor antagonist anakinra in a patient with ARDS caused by COVID‐19 infection: A case report. Clin Case Rep. 2020; 00: 1– 5. https://doi.org/10.1002/ccr3.3307