Description

Abstract

Our team applied synthetic biology to address cardiovascular diseases in Taiwan – a pressing issue accounting for 22.5% of deaths among the elderly aged 65 or above (Statista, 2022). Through research, we found that lumbrokinase, an enzyme extracted from Lumbricus bistamus (red earthworms), holds promise in dissolving fibrin in thrombi, which are responsible for blood clotting and strokes. However, there were two issues we found: the high cost and moral implications of current lumbrokinase markets, as well as medication non-compliance and high-fat dietary habits among elderly patients with cardiovascular diseases. To address the limitations of current lumbrokinase production methods, Worm Out Clots aims to establish improvements in two aspects: probiotic platform and industrial production. We hope to develop an innovative production pathway that enhances quality and lowers the price, while preventing mass killing of earthworms. On the other hand, develop a probiotic platform to address problems the elderly in Taiwan faced by creating an automated, in-vivo probiotic platform integrated with lumbrokinase.

Defining Problems

Since 2018, Taiwan has become an aging society, with the population aged 65 and above growing at an annual rate of 5%. In 2022, the median age in Taiwan is 41.3, compared to the global median age of 30.3, ranking Taiwan 27th globally in terms of median age (ASPS, 2022). This aging trend is accompanied by an increasing prevalence of age-related diseases. For instance, arteries in the elderly tend to lose flexibility, leading to a slower cardiovascular system and more delicate blood vessels. Consequently, thrombi can form in the vessels, potentially obstructing blood flow, causing blockages, and damaging organs due to the lack of oxygen transfer. Cardiovascular diseases resulting from blood clots affect 22.5% of the Taiwanese population aged 65 and above. Current treatment methods include medication, surgery, and cardiac rehabilitation. It is safe to say that Taiwan possesses one of the best medical care in the world, yet the mortality and incidence of CVD continues to rise due to lack of timely treatment.

Percentage of Taiwan's total population aged 65 or above based on 1960-2022. Statista, February 2023
Global cardiovascular diseases mortality rate out of 1000 by gender based on age. MDPI, April 2019

Thrombus Background

One of the primary causes of cardiovascular diseases is the formation of blood clots in veins or arteries. Normally, the coagulation mechanism produces a thrombus to achieve hemostasis when vessels experience rupture or injury. However, under certain conditions, a thrombus will recognize something else as a wound or invasive pathogen which triggers the binding process of platelets and fibrinogen. This clot can obstruct blood vessels, preventing the flow of blood, leading to a failure in the transfer of oxygen and nutrients. As a result, it causes cell or organ death.

The composition of a human thrombus is complex, comprising various substances such as platelets, cholesterol, fibrin, and erythrocytes. Among these, platelets and fibrin serve as the main structural components. During thrombus formation, fibrinogen is converted into fibrin, which then combine with substances like platelets and erythrocytes to form a large compound that blocks vessels.

Thrombus formation can be attributed to three primary factors: endothelial injury, which damages the inner lining of blood vessels; atrial fibrillation, a heart rhythm disorder that increases the risk of clot formation in the atria; and abnormal blood composition, where imbalances in blood components can promote clotting.

Endothelial Injury: This is primarily caused by aging, hyperlipidemia, and hypertension. These factors lead to the invasion of low-density lipoprotein cholesterol and the formation of atherosclerotic plaques. When these plaques rupture, the remaining cholesterol in the arterial wall oxidizes and attracts platelets. These platelets then signal the coagulation cascade, eventually causing fibrin molecules to combine and form long threads. These threads entangle the platelets, creating a spongy mass that gradually hardens and contracts to form a white thrombus. In more severe cases, this white thrombus may detach from the vessel wall, leading to emboli that can obstruct blood flow. This is commonly observed in cases of myocardial infarction.

Atrial Fibrillation and Abnormal Blood Composition: Blood flow disorders often arise from atrial fibrillation in the atrium. The irregular heart rhythm frequently leads to the formation of blood clots, particularly in the atria. Additionally, abnormal blood composition can result in increased blood viscosity, contributing to the development of deep vein thrombosis. Both factors play crucial roles in the formation of red thrombi by triggering the activation of blood coagulation factors. This activation process attracts fibrin, a protein involved in thrombus formation, resulting in red thrombus.

Furthermore, since these red thrombi can circulate within the system, they can lead to strokes originating from various parts of the body. Examples include ischemic necrosis of the intestine, where the thrombus travels to the large and small intestines, and pulmonary embolism, where a red thrombus originating in the femoral vein moves to the lungs.

Lumbrokinase

Recognizing that fibrin is a common component in various forms of thrombosis, our research team sought to identify proteins capable of dissolving it. This led us to discover lumbrokinase. Derived from Lumbricus bimastus (red earthworm), lumbrokinase belongs to the group of serine proteases. It has the ability to promote the fibrinolytic system by degrading natural fibrin found in the earthworm's body. Introduced by Mihara in 1991, lumbrokinase is utilized in enzyme formulations to dissolve proteins, playing a crucial role in thrombus removal. The enzyme, tissue-type plasminogen activator (t-PA), converts plasminogen (the inactive form of plasmin) into its active form, plasmin, thereby facilitating the breakdown of fibrin.

In blood vessels, thrombin converts fibrinogen into fibrin, which then aggregates with substances such as platelets and erythrocytes to form a thrombus. According to the principles of thrombus formation and the fibrinolytic system, if lumbrokinase can effectively dissolve both fibrinogen and fibrin and disrupt the thrombus structure, it may be possible to remove the blockage.

There are three main mechanisms of how Lumbrokinase remove the thrombus:
-Directly dissolves fibrinogen and fibrin in the blood vessels so that the thrombus will not be produced
-Promotes t-PA (endogenous tissue plasminogen activator) to produce plasmin, further destroying the thrombus
-Inhibits the collective and activation of platelets

Moreover, some studies suggest that lumbrokinase might have potential benefits in improving blood flow and reducing the risk of clot-related conditions such as hypertension, cardiovascular disease, brain infarction (stroke), and varicose veins.

Issues

1. Production process with low efficiency

Traditional methods of lumbrokinase extraction have focused on three specific earthworm species: Lumbricus rubellus, Eisenia fetida, and Lumbricus bimastus. These methods require DNA extraction from these earthworms, necessitating the culling of a significant number. Not only does this approach result in considerable loss of life due to its inefficiency, but it also incurs substantial labor and resource costs in the manufacturing process. Furthermore, products derived from this conventional extraction process are often contaminated with impurities, adversely affecting both their quality and efficacy.

2. Dietary Issues in Taiwan

Recent surges in obesity rates in Taiwan are primarily attributed to shifts in dietary patterns. High-calorie and high-fat foods have not only become prevalent in modern diets but are also increasingly favored by the public. The obesity rate among Taiwanese people reached a staggering 50.3% between 2017 and 2020 according to Statista of Taiwan Administration. Compared to other Asian countries with lower obesity rates, this alarming figure underscores the severity of the obesity problem and its significant threat to the health of the Taiwanese population.

Furthermore, according to a study conducted by Statista, the prevalence of hypertension has reached 27% and hyperlipidemia has reached 26% in Taiwan. Approximately one in four Taiwanese adults over the age of 18 suffers from hyperlipidemia, projected to an estimation of 5 million individuals. These findings highlight the severe impact that elevated levels of triglycerides and cholesterol are having on the lives of Taiwanese people, underscoring the urgent need for effective solutions to address the health challenges posed by high-fat dietary habits.

Our approach and solution

Recognizing the gravity of the health issues previously discussed, our team has formulated a dual-pronged strategy: first, to enhance the efficiency of current industrial lumbrokinase production, and second, to develop an automated in-vivo probiotic platform.

Initial Stage and Biotechnological Methods: In the initial phase of our experiment, we employed various biotechnological methods—including reverse transcription, transformation, and polymerase chain reaction (PCR)—to generate E. coli DH5α strains containing lumbrokinase sequences. Upon ligation of the PCR fragment with the pGEM-T EASY vector, we instituted multiple checkpoints. These included blue-white screening, small-scale restriction enzyme checks, and DNA sequencing to verify that the PCR fragment's sequence aligned with the lumbrokinase sequence.

IPTG Induction and Industrial Scale-Up: We subsequently implemented IPTG induction as part of our industrial production efforts. After identifying the correct clones with the lumbrokinase gene, we carried out a small-scale induction test using IPTG to trigger lumbrokinase production. Verification was performed through SDS-PAGE and blue-white screening to confirm successful lumbrokinase production in E. coli BL21 colonies post-IPTG induction.

Oleate Induction and In-Vivo Probiotic Platform: In parallel, our team designed and tested an oleate induction mechanism for the in-vivo probiotic platform. We adapted the pET-22b-LK vector, previously used in IPTG induction, for compatibility with oleate induction. Following vector modification, we selected the appropriate clones and initiated a small-scale induction test using fatty acids. The success of lumbrokinase production was subsequently verified through SDS-PAGE and blue-white screening.

Improvements in treatment

Current treatments for cardiovascular diseases, such as anticoagulants and thrombolytics, carry significant risks, including bleeding and bruising. Surgical interventions also come with their own set of risks, such as infection, bleeding, and further damage to blood vessels. Utilizing lumbrokinase, a natural protein extracted from earthworms, may offer a safer alternative by potentially mitigating some of these risks associated with existing treatments.

The range of applications for lumbrokinase is extensive, suggesting its potential efficacy in treating various cardiovascular issues. These include alleviating pain, enhancing wound healing, restoring limb functionality, breaking down blood clots, reducing pulmonary artery obstruction, improving oxygenation, restoring cerebral blood flow, and aiding in stroke recovery. Therefore, lumbrokinase has the potential to address a myriad of cardiovascular problems, thereby saving millions of lives.

Furthermore, the development of an in-vivo probiotic platform that synthesizes lumbrokinase could offer additional advantages. For instance, patients may not need to take this supplement daily, reducing the likelihood of non-compliance due to forgetfulness. This approach also provides a preventative measure for those unaware of their cardiovascular condition, thus addressing issues related to patient awareness.

Inspiration

At first, we thought of diabetes as a major issue and disease in Taiwan. Hyperglycemia triggers inflammation in blood vessels and attracts white blood cells to the inner arterial walls. Coupled with the deposition of low-density lipoprotein (commonly known as 'bad cholesterol'), this forms arterial plaque that hardens and narrows the vessels, potentially even blocking them. For diabetic patients, this necessitates regular insulin injections to lower blood sugar and clear these blockages. The long-term discomfort associated with these injections inspired the development of oral insulin, which in turn inspired our research approach. However, diabetes is just one facet of a broader health crisis in Taiwan. Despite significant investments in research, there are other pressing national health issues that require attention. One such issue is cardiovascular disease.

Cardiovascular diseases have consistently ranked among the top ten causes of death in Taiwan for several years. In fact, more people die annually from cardiovascular diseases than from cancer, which is the leading cause of death. To address this, we took inspiration from the oral insulin treatment for diabetes and sought to develop oral lumbrokinase. Our aim is to tackle two societal issues: high-fat dietary habits and medication non-compliance, particularly among the elderly who may forget to take their medication. No matter how effective a drug may be in treating cardiovascular diseases, it becomes irrelevant if patients forget to take it. To mitigate this issue, we plan to develop an automated in-vivo probiotic platform designed to extend the time intervals between required dosages, thus addressing the prevalent issue of medication non-compliance among the elderly.

References