Team:William and Mary/Description

Our Project Our Inspiration Our Project Goals How COVID-19 Impacted Our Project References


Project Description

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Our Project


What is TheraPUFA?

Our project comprises two aims:

  1. Design a novel “smart” nasal probiotic that secretes polyunsaturated fatty acids (PUFAs) for use as a broad-spectrum antiviral therapeutic
  2. Determine the feasibility of the probiotic through extensive, iterative mathematical modeling

Why develop a broad-spectrum antiviral therapy?

Combatting COVID-19 Requires:

The emergence of the novel coronavirus SARS CoV-2 has emphasized the urgent need for rapidly-developed vaccines, accurate diagnostic tools, and reliable therapeutics, including broad-spectrum antiviral drugs capable of combating a wide variety of viruses. Though targeted therapies are also critical, broad spectrum antiviral drugs can be effective against viruses that have yet to even emerge, thus constituting a means of pandemic preparedness.

Why develop a lipid-based therapy?

Lipids play a fundamental role in viral infection and the human immune response to viruses, warranting investigation into the potential of lipid-based antiviral treatments. Specifically, polyunsaturated fatty acids (PUFAs) have demonstrated antiviral effects against enveloped and positive strand RNA viruses, and regulate inflammation through their metabolites. To read an extensive literature review regarding the antiviral effects and metabolites of PUFAs, please visit our Engineering Success page.


Antiviral Properties of PUFAs

Why design a "smart" antiviral therapeutic?

Viruses such as SARS-CoV-2 cause a wide range of symptoms in patients that can change dramatically over the course of infection, necessitating the use of a smart drug such as TheraPUFA. For example, certain individuals remain completely asymptomatic while infected, whereas other individuals experience mild to severe inflammation. Extreme changes can be observed within a single patient, who may appear asymptomatic during a 5-6 day incubation period, then experience severe inflammation in the form of a cytokine form, followed by severe immunosuppression.


An ideal drug must “sense” indicators of inflammation and respond accordingly. Drugs that do not respond to inflammation cannot curb the damage it is capable of causing, whereas explicitly anti-inflammatory drugs (such as steroids) may suppress the immune response and hinder viral clearance, depending on when they are administered during infection.

How does TheraPUFA work?

TheraPUFA is a “smart” probiotic in that it can sense and suppress excess inflammation, which can occur during infection by SARS-CoV-2. TheraPUFA constitutively secretes arachidonic acid (AA), which has both pro- and anti-inflammatory metabolites, then switches to explicitly anti-inflammatory docosahexaenoic acid (DHA) in the case of excess inflammation, as indicated by high levels of pro-inflammatory cytokines. Both AA and DHA have demonstrated antiviral effects against enveloped and positive strand RNA viruses in vitro. Please visit our Design page to learn more about how TheraPUFA works to combat viral infection.

How is TheraPUFA novel?

TheraPUFA is novel in both probiotic design and the mathematical modeling employed to determine feasibility.

  • Lipid-based: Unlike prevalent RNA therapies, TheraPUFA utilizes PUFAS, a type of bioactive lipid, to combat infection. TheraPUFA provides a means to export PUFAS following their synthesis within a bacterial cell, which has not been previously characterized.

  • Smart: TheraPUFA comprises a “smart” drug in that it can sense and suppress excess inflammation, which can occur during infection by SARS-CoV-2 and similar viruses. The ability to respond dynamically and intelligently to different degrees of inflammation distinguishes TheraPUFA from standard pharmaceuticals such as Dexamethasone and Remdesivir.

  • Nasal: TheraPUFA is a nasal probiotic, rather than a more traditional gut probiotic. Within the nasopharynx, a dominant site of early infection by SARS-CoV-2, TheraPUFA can protect vulnerable cells with high expression of ACE-2 receptors, and prevent spread of infection into the lungs when administered prophylactically (Hou et al., 2020, Cell).

  • Stochastic, Spatially-Conscious Model: To investigate the feasibility of TheraPUFA, we constructed a complex model that simulates the probiotic’s effect on viral load and cytokine concentrations. Our model extends beyond pre-existing probiotic models by accounting for transcriptional stochasticity and spatial heterogeneity within the nasal cavity. See the results of the model.

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Our Inspiration




Prior to the COVID-19 pandemic, we at William & Mary iGEM explored a variety of potential project topics, ranging from non-hormonal contraceptives to artificial meat. Though our ideas differed widely, spanning over half of the thirteen iGEM tracks, all projects proposed to apply synthetic biology to a pressing global challenge.

In early February, it became clear that no challenge was more urgent or more widespread than the pandemic itself. We witnessed the toll of the virus world-wide, concerned especially for our teammates’ families in China. As time passed, the pandemic inevitably reached our local community. A local ICU nurse recounted her experience caring for COVID patients to us, stating that hospitalization due to the virus is “terrifying” for patients. “There’s a lot of fear in people’s eyes,” she said. She described the discomfort and isolation her patients faced. Joining researchers around the world in the concerted effort to overcome SARS-CoV-2, we unanimously decided to pursue a COVID-19 related project.

Academic researchers, pharmaceutical companies, and government agencies have focused their united efforts into vaccines and antiviral therapies. However, vaccines can take months to years to develop. Furthermore, though some broad-spectrum antivirals have shown promise, they vary in their efficacy.

Reliable broad-spectrum antiviral therapies are necessary in the treatment of viral infections, especially when vaccines or other specific therapies are unavailable. Broad spectrum antiviral therapies could be effective against viruses that have yet to even emerge, contributing to pandemic preparedness. Prior to designing our own broad spectrum antiviral therapy, we researched pre-existing therapeutics currently available for the treatment of COVID-19. We investigated with particular attention the ability, or inability in some cases, of these therapeutics to respond to varying degrees of inflammation.

We investigated the following proposed broad-spectrum antiviral therapies prior to designing TheraPUFA:

  • Chloroquine (CQ) and Hydroxychloroquine (HCQ): In February and March, clinical trials and in-vitro studies in China suggested that HCQ was effective in combating infection by SARS-CoV-2 (Alia & Grant-Kels, 2020, J Am Acad Dermatol.). Their findings were supported by a Gautret et al., a small study that documented reduction in mortality correlated with treatment with HCQ and azithromycin (2020, Int. J. Antimicrob. Agents). However, studies with larger sample sizes published by New England Journal of Medicine could not establish a prophylactic or therapeutic effect of HCQ against SARS-CoV-2 infection (Geleris et al., Boulware et al., 2020). Furthermore, significant side effects led the FDA to caution against using HCQ outside of a clinical trial, even in the case of emergency use authorization (EUA).

  • Remdesivir: Patients treated with Remdesivir experienced a shortened recovery duration by a mean time of four days, according to a New England Journal of Medicine study (Beigel et al., 2020). Though a lower rate of mortality was also observed, this observation was not statistically significant. Later, a WHO Solidarity trial in preprint found that neither Remdesivir nor HCQ reduced mortality (Pan et al., 2020, medRxiv preprint).

  • Dexamethasone: Among patients given respiratory support, such as ventilation or oxygen, patients treated with the glucocorticoid dexamethasone exhibited a lower 28-day mortality than patients given usual care. However, among patients given no respiratory support, a slight increase in mortality was observed in patients who received dexamethasone compared to patients who received usual care (The RECOVERY Collaborative Group, 2020, New Engl. J. Med.). These results may suggest that the efficacy of dexamethasone depends on the stage of infection or inflammation.

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Our Project Goals



We sought to determine the feasibility of our probiotic design by reviewing literature, consulting experts and stakeholders and mathematically modeling our circuits. We aimed to:

  • Conduct a literature review on the antiviral effects of PUFAs.

  • Identify feasible genetic parts for PUFA synthesis and export in a prokaryotic chassis.

  • Conduct a literature review on commensal bacterial species within the nasopharyngeal microbiome. Identified species that could be safely engineered for use as a nasal probiotic.

  • Consult experts and stakeholders, specifically medical professionals who would administer the therapy if it is implemented in the future. Requested their feedback on the design of our project in order to make our proposed therapy as safe, effective, and appealing/easy-to-use as possible.

  • Consult experts in probiotics and drug manufacturing for guidance on the implementation of our potential probiotic, and on strategies to make the probiotic as accessible as possible.

  • Utilize mathematical modeling to determine whether our design is feasible. This included modeling the synthesis and export of PUFAs, as well as their interactions with viruses and human cells.

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How COVID-19 Impacted Our Project

While COVID-19 motivated our entire project, it also impacted our work in a variety of other ways. COVID-19 prevented us from accessing our lab on campus at William & Mary, directing us to choose a project that focused on challenging design and rigorous mathematical modeling that would have been impossible in a regular wet lab season. Furthermore, COVID-19 eliminated opportunities to communicate and work in-person. Despite these limitations, the completely remote format empowered us to pursue an extensive literature review, encompassing over 250 research papers studied, and devote our focus to a rigorous mathematical model. Over the course of the project, we ran over 220 simulations, and investigated the effects of 11 different parameters. Below is a brief list of changes we made to continue our work remotely:

  • Hosted team meetings via Zoom

  • Emphasized platforms such as Benchling, Discord, Trello and email to communicate goals and progress

  • Utilized workspaces that allowed for collaboration and simultaneous editing, such as Google CoLaboratory and Google Documents

  • Corresponded with stakeholders via email, phone, or Zoom

  • Hosted the iGEM 2020 MidAtlantic Meetup via Zoom, inviting teams and speakers to present virtually

  • Participated in outreach and education events virtually, presenting over Zoom to students in middle school and highschool.

  • Collaborated virtually with other teams, participating in a podcast with Pittsburgh iGEM and a video series with Purdue iGEM

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References



Alia, E., & Grant-Kels, J. M. (2020). Does hydroxychloroquine combat COVID-19? A timeline of evidence. Journal of the American Academy of Dermatology, 83(1), e33–e34. https://doi.org/10.1016/j.jaad.2020.04.031

Beigel, J. H., Tomashek, K. M., Dodd, L. E., Mehta, A. K., Zingman, B. S., Kalil, A. C., Hohmann, E., Chu, H. Y., Luetkemeyer, A., Kline, S., Lopez de Castilla, D., Finberg, R. W., Dierberg, K., Tapson, V., Hsieh, L., Patterson, T. F., Paredes, R., Sweeney, D. A., Short, W. R., Touloumi, G., Lye, D. C., Ohmagari, N., Oh, M.-d., Ruiz-Palacios, G. M., Benfield, T., Fätkenheuer, G., Kortepeter, M. G., Atmar, R. L., Creech, C. B., Lundgren, J., Babiker, A. G., Pett, S., Neaton, J. D., Burgess, T. H., Bonnett, T., Green, M., Makowski, M., Osinusi, A., Nayak, S., & Lane, H. C. (2020). Remdesivir for the Treatment of Covid-19 — Final Report. New England Journal of Medicine. https://doi.org/10.1056/NEJMoa2007764

Boulware, D. R., Pullen, M. F., Bangdiwala, A. S., Pastick, K. A., Lofgren, S. M., Okafor, E. C., Skipper, C. P., Nascene, A. A., Nicol, M. R., Abassi, M., Engen, N. W., Cheng, M. P., LaBar, D., Lother, S. A., MacKenzie, L. J., Drobot, G., Marten, N., Zarychanski, R., Kelly, L. E., Schwartz, I. S., McDonald, E. G., Rajasingham, R., Lee, T. C., & Hullsiek, K. H. (2020). A Randomized Trial of Hydroxychloroquine as Postexposure Prophylaxis for Covid-19. New England Journal of Medicine, 383(6), 517-525. https://doi.org/10.1056/NEJMoa2016638

Center for Drug Evaluation and Research. (2020, July 1). FDA cautions use of hydroxychloroquine/chloroquine for COVID-19. Retrieved October 17, 2020, from https://www.fda.gov/drugs/drug-safety-and-availability/fda-cautions-against-use-hydroxychloroquine-or-chloroquine-covid-19-outside-hospital-setting-or

Dexamethasone in Hospitalized Patients with Covid-19 — Preliminary Report. (2020). New England Journal of Medicine. https://doi.org/10.1056/NEJMoa2021436

Gautret, P., Lagier, J. C., Parola, P., Hoang, V. T., Meddeb, L., Mailhe, M., Doudier, B., Courjon, J., Giordanengo, V., Vieira, V. E., Tissot Dupont, H., Honoré, S., Colson, P., Chabrière, E., La Scola, B., Rolain, J. M., Brouqui, P., & Raoult, D. (2020). Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial. International journal of antimicrobial agents, 56(1), 105949. https://doi.org/10.1016/j.ijantimicag.2020.105949

Geleris, J., Sun, Y., Platt, J., Zucker, J., Baldwin, M., Hripcsak, G., Labella, A., Manson, D. K., Kubin, C., Barr, R. G., Sobieszczyk, M. E., & Schluger, N. W. (2020). Observational Study of Hydroxychloroquine in Hospitalized Patients with Covid-19. New England Journal of Medicine, 382(25), 2411-2418. https://doi.org/10.1056/NEJMoa2012410

Hou, Y. J., Okuda, K., Edwards, C. E., Martinez, D. R., Asakura, T., Dinnon, K. H., 3rd, Kato, T., Lee, R. E., Yount, B. L., Mascenik, T. M., Chen, G., Olivier, K. N., Ghio, A., Tse, L. V., Leist, S. R., Gralinski, L. E., Schäfer, A., Dang, H., Gilmore, R., Nakano, S., … Baric, R. S. (2020). SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract. Cell, 182(2), 429–446.e14. https://doi.org/10.1016/j.cell.2020.05.042

Pan, H., Peto, R., Abdool Karim, Q., Alejandria, M., Henao Restrepo, A. M., Hernandez Garcia, C., Kieny, M. P., Malekzadeh, R., Murthy, S., Preziosi, M.-P., Reddy, S., Roses, M., Sathiyamoorthy, V., Rottingen, J.-A., & Swaminathan, S. (2020). Repurposed antiviral drugs for COVID-19; interim WHO SOLIDARITY trial results. medRxiv, 2020.2010.2015.20209817. https://doi.org/10.1101/2020.10.15.20209817