Abstract
Coronary artery disease (CAD) accounts for 17.8 million deaths worldwide annually, with especially high rates in the southeastern United States. Current detection methods are costly and invasive, making them inaccessible to many. CADlock utilizes microRNA (miRNA) biomarkers to provide a novel screening tool for CAD. Our project uses padlock probes with rolling circle amplification/transcription to quantify miRNAs via fluorescence. Furthermore, we developed ProbeBuilder, a padlock probe generator software based on miRNA input, and Micro-Q, a portable fluorescent quantifier that allows for point-of-care screening for CAD. Current miRNA research in CAD is limited, which led us to offer CADlock as a tool for researchers to add miRNA characterization. We contacted stakeholders, including the American Heart Association, Georgia Office of Cardiac Health, researchers, cardiologists, and patients to gain feedback on the impact of our project. CADlock provides an additional detection method for researchers and physicians, facilitating a point-of-care screening procedure for CAD (see Fig. 1).
Defining the Problem
Coronary artery disease (CAD) is the third leading cause of death globally, and the leading cause of death in the United States (Brown et al., 2022). Within the U.S., the problem is more prevalent in the southeastern region of the United States, including our home state of Georgia (Centers for Disease Control and Prevention [CDC], 2020).
Pathophysiology
CAD is caused by the development of atherosclerotic plaque, a build-up of fatty material, cholesterol, cellular waste products, calcium, and fibrin, which can cause a narrowing of the arteries to block blood flow (Shahjehan & Bhutta, 2022). This blockage occurs in a three step process (see Fig. 2):
- Buildup: Subendothelial deposits of foam cells (lipid macrophages) break the tunica intima - the innermost layer of the heart, forming a “fatty streak” (Shahjehan & Bhutta, 2022).
- Development: Low-density lipoprotein particles and foam cells activate T cells, which result in immune responses that nourish the plaque, making it increase in size, a process called angiogenesis (Baixeras et al., 2014).
- Blockage: The atheroscleoric plaque continues to increase in size, calcifying over time and narrowing the heart artery in a process called atherostenosis (Baixeras et al., 2014). This process causes occlusion in the coronary arteries, reducing vital blood supply to the heart (Centers for Disease Control and Prevention [CDC], 2020). A 100% occlusion in arteries results in death of heart tissue, ischemia, leading to a myocardial infarction (heart attack) (Shahjehan & Bhutta, 2022).
Symptoms
Coronary artery disease (CAD) can come in a variety of symptoms, some more obvious than others (Center for Disease Prevention [CDC], 2020). Symptoms are usually presented differently by gender, with women often presenting symptoms 7 to 10 years later than men (see Fig. 3) (Maas & Appelman, 2010). These symptoms include but are not limited to:
- Angina: Chest pain with pressure or tightness, usually a brief, stabbing pain.
- Shortness of Breath: Intense tightening in chest with difficulty breathing and breathlessness.
- Fatigue: Increased tiredness, the heart can’t pump blood to meet body’s needs.
- Nausea: Increased lightheadedness accompanied with dizziness and cold sweats.
Current Diagnostics
There are several diagnostics used by physicians to diagnose coronary artery disease (see Fig. 4) (Mayo Clinic, 2022):
- Electrocardiogram (EKG): Measures the electrical activity of the heart, electrodes are placed on the chest to record the heart’s electrical signals, helping detect arrhythmias (irregular heartbeats).
- Echocardiogram (ECG): Uses soundwaves to create pictures of the beating heart, can visualize how blood moves through the heart vessels.
- Nuclear Stress Test: A radioactive tracer is placed via an Intravenous (IV) Injection during an EKG, allowing clearer images to be seen of blood flow through the heart.
- Computed Tomography Angiogram (CT) Scan: Displays calcium deposits and blockages in arteries; Requires exposure to radiation through a CT Scanner via an IV injection.
Lambert iGEM aims to provide a lower-cost, point-of-care screening tool for CAD using microRNA (miRNA) biomarkers linked to CAD, intended to complement diagnostics mentioned above. While genetic factors and family history cannot be controlled or changed by a patient, other lifestyle factors can be changed. For this reason, we aim to make information about cardiovascular disease and synthetic biology more accessible and inclusive to the public, especially for historically underserved populations.
We determined the cost per reaction by dividing the bulk cost by the amount of reactions per kit, resulting in a cost of $14.59 per reaction (excluding capital equipment costs) (see Table 1). Including capital equipment (reusable items or tools) such as Micro-Q, OpenCellX, instructions, gloves, goggles, and beakers, the cost per reaction increases to $38.96 (see Table 1). To estimate the cost of bioengineered components such as the padlock, flurophore, and quencher probes, we estimated the total number of reactions we were able to do based on our experience with the tools. The average cost for current CAD detection methods such as angiograms and lipid panels cost about $1363 and $156 respectively. With a cost per reaction under $40, our kit provides consumers with a cost-effective and efficient way to measure a user's risk for CAD.
| Item | Cost per Reaction (Without Capital Equipment) | Cost per Reaction (With Capital Equipment) |
|---|---|---|
| Hardware | $0.00 | $21.37 |
| miRNA Biomarker Detection System | $9.85 | $9.85 |
| Other Materials | $0.57 | $3.57 |
| Total Costs | $14.59 | $38.96 |
microRNA Background
miRNAs are small, non-coding RNA molecules that regulate gene expression via translational repression or cleavage of target mRNAs. Most miRNAs are transcribed from DNA sequences, which can then be transcribed into primary, precursor, and mature miRNAs (O‘Brien et al., 2018). miRNAs interact with the 3’UTR region of mRNAs to induce translation repression or mRNA deadenylation/decapping, making mRNAs susceptible to degradation, affecting gene expression (see Fig. 5) (O‘Brien et al., 2018).
The expression of miRNAs is often altered in diseases, including cardiovascular diseases such as coronary artery disease (CAD). The use of microRNAs (miRNAs) as biosensors for CAD is a novel approach that could improve the diagnosis and management of CAD. miRNA profiling can be used to detect CAD at an early stage, when it is still asymptomatic and before any cardiovascular event occurs (Kaur et al., 2020). The diagnosis of CAD remains primarily clinical, which makes it difficult to predict the prognosis or assess the risk of future cardiac events. Furthermore, patients with angina pectoris or myocardial infarction are often misdiagnosed due to their non-specific symptoms. In this scenario, the use of miRNA profiling could be beneficial because it represents an effective tool for detecting hidden pathologies.
5p vs 3p Structure of miRNAs:
Many miRNAs are identified by their 5p and 3p positions. The 5p position is present in the forward (5’ - 3’) position and the 3p strand is present in the reverse position (3’ - 5’) (McAlinden, 2015).
CAD miRNA Selection:
After careful literature review, we decided to design biosensors to target hsa-miR-1-3p and hsa-miR-133-3p as biomarkers for screening and monitoring of coronary artery disease. In particular, these two biomarkers are upregulated in CAD patients (see Fig. 6) (Xiao et al., 2019). We specifically avoided downregulated miRNAs to ensure that our sensors would be able to detect these small concentrations of miRNAs.
- hsa-miR-1-3p: This miRNA is associated with Gene Ontologies: GO: 0010460 and GO: 0010614, which regulates the hypertrophy (growth) of cardiac muscle (miRBase).
- hsa-miR-133a-3p: This miRNA is associated with Gene Ontologies: GO: 0010989 and GO: 0010614, which are associated with negative regulation of lipoproteins, which are important in plaque formation. miR-133a-3p also plays a crucial role in cardiomyocyte proliferation.
Additionally the following miRNAs will be used to measure the progression of CAD within patients (miRBase).
- hsa-miRNA-122-5p: This miRNA is associated with Gene Ontologies GO:0005615 and GO: 0010614 associated with reduced fatty acid metabolism in atherosclerotic development.
- hsa-miRNA-126-3p: This miRNA is associated with Gene Ontologies: GO:0045766 and GO:1903589, which are associated with blood vessel endothelial proliferation, which contribute to plaque growth.
- hsa-miRNA-150-5p: miRNA is associated with Gene Ontologies: GO:0090050 and GO:1905562, which are associated with angiogenesis and vascular cell proliferation.
miRNA researchers, Dr. Searles and Dr. Delles, provided additional evidence that our selections were worthwhile.
Lambert iGEM also used hsa-miR-451a as a control miRNA due to the difference of miRNA levels in populations and sampling locations (Huang et al., 2011). hsa-miR-451 is not impacted by the pathogenesis of coronary artery disease, and thus serves as a benchmark for varying miRNA levels (Mussbacher, 2020).
Our Approach
Lambert iGEM’s CADlock utilizes a multifaceted approach to address difficulties in the screening and diagnosis of coronary artery disease (CAD). We developed biosensors for microRNA (miRNA) biomarkers (hsa-miR-1-3p & hsa-miR-133a-3p) associated with CAD in conjunction with Micro-Q, a frugal fluorescence quantification device.
Our biosensors use padlock probes along with rolling circle amplification to detect and amplify microRNA's (miRNA) to be quantified by their fluorescence. We tested both rolling circle transcription and rolling circle amplification with several reporter mechanisms, including Lettuce aptamers, Spinach aptamers, and linear DNA probes. However, Lambert iGEM faced difficulty while designing the padlock probes due to a lack of software to automate the process. For this reason, Lambert iGEM designed ProbeBuilder, a software that can generate padlock probes based on the target miRNA sequence and the desired reporter sequence.
Throughout our project, Lambert iGEM consulted stakeholders such as patients, cardiologists, researchers, and organizations that work in healthy eating and cardiovascular disease research and advocacy. Since cardiovascular disease disproportionately affects those from marginalized groups, we took several initiatives to address these inequalities, including a cookbook with heart-healthy recipes from many different cultures and a communication board for nonverbal patients to communicate with their doctors. In addition, we created several education initiatives to spread awareness about heart-healthy habits and synthetic biology.
With CADlock, Lambert iGEM holistically addresses the difficulties of diagnosis of coronary artery disease through a variety of approaches including our miRNA biosensors, frugal hardware, and educational initiatives.