Overview
The project of 2021 NYCU-Taipei iGEM Team—“NATTO IT OUT”, presents a gene-edited E. coli Nissle 1917 (EcN) product that could produce Nattokinase and could also be remote-controlled by an optogenetic system. Nattokinase has proven to be a good thrombolytic agent; therefore, it could serve as a promising supplement to prevent cardiovascular diseases [1, 2, 3]. Our project specifically targets groups at high risk of deep vein thrombosis (DVT). Our project “NATTO IT OUT” aims to “use Nattokinase to wipe out thrombosis”.
Background
"NATTO IT OUT" aims to carry out an effective prevention method for cardiovascular diseases, the top cause of death worldwide, especially deep vein thrombosis (DVT).
Deep vein thrombosis is developed from blood clots in the veins of lower limbs, causing pain, swelling, tenderness, redness, warmth, and impeding of the blood flow. The swelling compresses the arteries, hindering the transport of oxygen and nutrition. This causes tissue necrosis, putting patients at risk of amputation. DVT can develop fatal complications, such as pulmonary embolism (PE). PE occurs when blood clots partially or totally detach from the vascular wall and be carried to lung arteries. It lodges the bloodstream of the lungs, resulting in damage of the lungs or other organs [4, 5]. According to statistics, “Of all PE patients, 25% would die suddenly, 23% would die within 3 months, 30% would die after 6 months, and the overall mortality rate is 37% 1 year after being diagnosed” [6]. Thus, DVT is a disease that couldn’t be ignored.
Nattokinase is a serine protease extracted from the Japanese traditional food Natto, which is processed from fermenting soybeans with B. subtilis. It has strong fibrinolytic ability, breaking down thrombi and fibrin by cleavage of cross-linked fibrin [2, 7]. Hence, studies have shown that it could lower the risk of thrombosis. Moreover, it is also demonstrated that Nattokinase has antihypertensive, anti-atherosclerotic, lipid-lowering, antiplatelet, and neuroprotective effects, which makes it a hopeful preventive drug for multiple cardiovascular diseases [1]. Our project mainly focuses on its thrombolysis effect on DVT. As a natural supplement, Nattokinase barely has adverse effects compared to medicines used to treat deep vein thrombosis, such as Heparin, Warfarin, and Rivaroxaban [8, 9].
Approach
Detection
Despite the severity of deep vein thrombosis and its complications, there still isn’t a convenient and handy device for people with high risk of thrombosis to test at home, anytime. Therefore, NYCU iGEM team developed a compact system, which detects the d-dimer in one’s saliva.
D-dimer, a product derived from the degradation of thrombi, is clinically used for initial detection of deep vein thrombosis risk [10, 11]. We developed a home-aid testing kit to test the absorbance intensity of d-dimer in one’s saliva; next, the absorbance intensity signal would be converted into the concentration of d-dimer in the saliva and sent to an app. After the signal was collected, the person’s thrombosis risk and recommended dosage of Nattokinase would be calculated and displayed on the app.
Prevention
To prevent deep vein thrombosis, one has to be “active”, and exercise their limbs more often. Even so, people with higher risk of thrombosis are mostly bedridden patients and the elderly.
In addition, studies have shown that if a person stops the treatment for unprovoked thrombosis for 6 months, the re-occurrence of thrombosis is approximately 20% for the first 4 years and 30% for 10 years [12]. Hence, it is important for those who have developed unprovoked thrombosis, and those with high risk of deep vein thrombosis to take drugs on a regular basis. To make this simpler, we devised E. coli live biotherapeutic products (LBP) that could consistently produce Nattokinase in the small intestine. In this way, there would be constant release of Nattokinase in the intestine of those who take our product.
With synthetic biology, we designed a gene-edited E. coli Nissle 1917 that could produce production-improved Nattokinase to help prevent the risk of deep vein thrombosis; additionally, combined with BphP1-QPAS1 light-inducible system, the production of Nattokinase could be remote-controlled through the previously mentioned app. The E. coli Nissle 1917 product would be lyophilized (freeze-dried) to ensure long term storage and sent to the small intestine with an enteric-coated HPMC capsule. After the capsule dissolves, the E. coli Nissle 1917 product would be released into the small intestine. The integration of OmpA-FimH fusion protein helps the E. coli product adhere to the epithelial cell of the small intestine, so that the E. coli product could form a colony inside the small intestine and constantly release Nattokinase. Finally, to guarantee the biosafety of our product, we adapted the MazEF toxin-antitoxin system to be our killswitch.
Inspiration
We started to pay attention to thrombosis after learning that in the COVID-19 pandemic world nowadays, up to one third of COVID-19 patients suffer from venous thromboemboli (VTE) [13]. Researchers have also suggested that thrombosis has been a common pathology to cardiovascular diseases [14]. Among VTE diseases, deep vein thrombosis (DVT) has gained less public attention yet it could be lethal.
Moreover, it is important for those at high risk of deep vein thrombosis to check their thrombosis status on a regular basis, since the symptoms are often mild and could be neglected. Taking preventive supplements consistently is also effective in preventing deep vein thrombosis. Thus, NYCU-Taipei iGEM team aims to offer a comprehensive approach to prevent DVT.
Throughout our iGEM journey, we not only provide a convenient thrombosis detection kit and genetically modified bacteria, but also hope to arouse public awareness of the danger of deep vein thrombosis. Besides modifying our designs, going through trial and errors, modelling, constantly discussing about our project, seeking for experts’ advice and public preferences, we have also exchanged our ideas with other iGEMers, promoted the knowledge of deep vein thrombosis, and shared what synthetic biology is to high school students. We hereby present to you NATTO IT OUT!
References
[1] Chen, H., McGowan, E. M., Ren, N., Lal, S., Nassif, N., Shad-Kaneez, F., Qu, X., & Lin, Y. (2018). Nattokinase: A Promising Alternative in Prevention and Treatment of Cardiovascular Diseases. Biomarker insights, 13, 1177271918785130. https://doi.org/10.1177/1177271918785130
[2] Kurosawa, Y., Nirengi, S., Homma, T. et al. A single-dose of oral nattokinase potentiates thrombolysis and anti-coagulation profiles. Sci Rep 5, 11601 (2015). https://doi.org/10.1038/srep11601
[3] Weng, Y., Yao, J., Sparks, S., & Wang, K. Y. (2017). Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease. International journal of molecular sciences, 18(3), 523. https://doi.org/10.3390/ijms18030523
[4] Lewis, S. (2020). Is Deep Vein Thrombosis an Emergency? https://www.healthgrades.com/right-care/vascular-conditions/is-deep-vein-thrombosis-an-emergency
[5] Johns Hopkins Medicine. https://www.hopkinsmedicine.org/health/conditions-and-diseases/pulmonary-embolism
[6] Cunha, JP. (2020). Can You Die if You Have a Blood Clot in Your Leg? https://www.emedicinehealth.com/ask_die_if_you_have_a_blood_clot_in_your_leg/article_em.htm#ask_a_doctor
[7] Kurosawa, Y., Nirengi, S., Homma, T., Esaki, K., Ohta, M., Clark, J. F., & Hamaoka, T. (2015). A single-dose of oral nattokinase potentiates thrombolysis and anti-coagulation profiles. Scientific reports, 5, 11601. https://doi.org/10.1038/srep11601
[8] Drugs.com. https://www.drugs.com/npp/nattokinase.html
[9] NHS website. https://www.nhs.uk/conditions/deep-vein-thrombosis-dvt/
[10] J. I. Weitz, J. C. Fredenburgh and J. W. Eikelboom. Journal of the American College of Cardiology 2017 Vol. 70 Issue 19 Pages 2411-2420. DOI: https://doi.org/10.1016/j.jacc.2017.09.024
[11] Pulivarthi, S., & Gurram, M. K. (2014). Effectiveness of d-dimer as a screening test for venous thromboembolism: an update. North American journal of medical sciences, 6(10), 491–499. https://doi.org/10.4103/1947-2714.143278
[12] North American Thrombosis Forum (NATF) https://natfonline.org/patients/what-is-thrombosis/
[13] Loo J, Spittle DA, Newnham M. COVID-19, immunothrombosis and venous thromboembolism: biological mechanisms. Thorax 2021;76:412-420.
[14] Raskob GE, Angchaisuksiri P, Blanco AN, Buller H, Gallus A, Hunt BJ, Hylek ER, Kakkar A, Konstantinides SV, McCumber M, Ozaki Y, Wendelboe A, and Weitz JI. Thrombosis A Major Contributor to Global Disease Burden. ATVR 2014; 34(11): 2363-2371.
