MIT: Mammalian Circuitry for Treatment of COVID-19 Cytokine Storms
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Much of COVID-19 pathology is still being unraveled; remotely, through the power of computational experimentation, we saw the opportunity to contribute with minimal risk. We thus studied literature pointing to unique biomarker patterns found in COVID-19 cytokine storms (CCSs), as well as underlying mechanisms of other overactive immune disorders, such as rheumatoid arthritis and psoriasis. Based on statistical analyses by Yang et al., IP-10 and MCP-3, two cytokines secreted by the IFN-γ cascade, when present together in elevated concentration, served as promising biomarkers for severe CCSs.
Additionally, quantifiable levels of cytokines and other immune signatures vary greatly between patients’ unique health profiles. Here, we realized a critical need: mitigation of the inflammatory response, catered to the individual patient’s immune response to SARS-CoV-2 infection. This challenge was one we believed synthetically engineered mammalian cells were well suited for: their ability to continually monitor state, implement dosage-feedback regulation, and utilize native biological parts pose many advantages over traditional pharmaceuticals.
Synthetic biology requires the engineering of nonlinear biology into predictable, digital behaviors; our goal was to employ effective digital molecular mechanisms to construct an analog, or “graded”, response which is tailorable to individual patients using already available synthetic biology-designed tools. Such an approach would ensure maintenance of systemic immune homeostasis.
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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.
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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.
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.
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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.
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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