Overview
Lambert iGEM’s 2022 project, CADlock, was shaped by the expertise of stakeholders during every step of its development. Inspired by the devastating impacts of coronary artery disease (CAD) and the growing research about microRNA (miRNA) biomarkers, we developed biosensors that can detect miRNA biomarkers correlated with CAD. In addition, we developed Micro-Q, a frugal fluorometer that quantifies fluorescence output from our biosensors (see Hardware). Throughout our project, we looked for ways to increase the accessibility of CAD diagnosis and expand the goals of healthy living to many different groups of people.
Initially, we intended our project to inform patients directly, but we discovered that the interpretation of results was best conducted by a trained medical professional. While we did discuss our project with patients diagnosed with CAD, we also contacted cardiologists, who could provide a clinical perspective on diagnosis and treatment. Many of the cardiologists we spoke to were hesitant about the feasibility of utilizing miRNA biomarkers for screening due to their relative novelty. In response to this, we contacted leading researchers in miRNA biomarkers to discuss what could be done to increase the potential for miRNAs to be used as a biomarker in clinical settings. Finally, we contacted community organizations that deal with factors that impact heart disease, including local food banks and the Georgia Office of Cardiac Care.
Patients
Throughout our project, we focused the development of CADlock on its end users: patients and cardiologists. However, due to safety and anonymity concerns, we faced some issues in speaking to patients. Eventually, we surveyed several individuals over the age of 18. All of the patients were made aware that participation was voluntary and they freely consented to participate in the surveys. In addition, they could discontinue the survey at any point or decline to answer any questions if they did not feel comfortable answering. We conducted our surveys through in-person interviews and asked about their demographic information, diagnosis story, and expectations/knowledge regarding CAD.
A majority of the patients we interviewed were middle-aged and received their diagnosis after their 40th birthday. Many of them were aware of their risk factors (such as family history, diet, and smoking) before their diagnosis; however, they were not concerned about heart disease and most did not take any additional precautions until symptoms worsened their quality of life. When we described the biosensors we developed, the patients expressed interest in using these tools to monitor their risk and/or condition both before and after diagnosis.
Based on this feedback, we were able to validate that patients who had no symptoms before diagnosis were interested in using our biosensors for post-diagnosis monitoring. Additionally, we discovered that patients who were aware of their risk factors for CAD such as family history were also interested in ongoing monitoring for an earlier diagnosis.
Cardiologists
As we investigated the feasibility of implementing CADlock biosensors as a point-of-care device in coronary artery disease (CAD) testing, we also spoke to several different cardiologists to understand their perspectives on the potential clinical use of these biosensors. Their feedback fueled our approach to further work in ensuring the future use of our biosensors and the design of Micro-Q. In most cases we held multiple meetings with each expert so they could follow our progress and contribute further feedback.
Although we allowed discussions to flow based on each cardiologist’s area of expertise, we created a list of general questions to lead each conversation. These are as follows:
- Have you heard of using miRNAs to diagnose cardiovascular disease?
- What are the benefits of early diagnosis for CAD/ischemic heart disease?
- What are current trends in the diagnosis of cardiovascular disease?
- What would be the most convenient way to use our biosensors in a doctor’s office? - - What type of equipment is available to you?
- How are patients diagnosed with coronary artery disease?
- What does a typical diagnosis story look like?
- What equipment and tests are typically used in diagnosing a patient with CAD?
- What conditions do you rule out before diagnosing a patient with CAD?
- Is there a need for a screening tool for CAD? What kind of patients would you screen?
Dr. Kavin Ezhilnambi
Dr. Kavin Ezhilnambi, a cardiologist in Tamil Nadu, India, strongly emphasized the importance of a highly sensitive, noninvasive, and small device for diagnosing cardiovascular diseases. Moreover, he expressed concern for accurate and precise lab equipment due to the constant presence of false positives and the costs of his measurement devices. Some of these techniques, including coronary angiograms, are useful in some cases but present side effects like radiation that deter patients from positively accepting these methods for diagnosis. Therefore, he supported our idea of creating a biosensor for monitoring the progression of CAD and as a screening device. He also suggested using standardized reference levels for the miRNAs: if the device is too sensitive (misinterprets the fluorescence reading), it may result in false positives that can cause patient anxiety. In addition, he encouraged us to look into the potential use of these biosensors after a patient has been diagnosed to monitor the progression of the disease.
Dr. Ugochukwu Egolum
Dr. Ugochukwu Egolum is a cardiologist specialist in Gainesville, Georgia. He had previously heard of miRNAs being used to diagnose acute myocardial infarction (MI) hours or days before the actual MI occurred. Therefore, he emphasized the importance of an early screening device, or a diagnostic tool like CADlock, so that similar conditions can be identified and eliminated before it progresses. He suggested that our device could be implemented at an Emergency Room (ER) or small urgent care clinics for concurrent use with both blood tests for troponin levels and cardiac CT scans to detect the miRNAs in the blood and plaque build-up in the arteries. However, since every patient presents a different set of symptoms or pain tolerance, he advised us to factor in these characteristics so that they can receive a specialized series of tests rather than a standardized scan. Even though he said our device may not replace current tests, its potential sensitivity level could complement current methods.
Dr. Omar Kashlan
Dr. Omar Kashlan, a cardiologist at a metro-Atlanta hospital, supported our early diagnosis approach in screening but held a different perspective on its benefit: patients immediately respond by adopting preventative lifestyle measures. By showing patients that they could have CAD, he argued that patients would be more likely to adopt healthier habits. He also mentioned how a calcium score, which is typically used in diagnosis, may not always be visibly abnormal, so having miRNAs as a biomarker will increase the accuracy of the screening tool. Like Dr. Egolum, he highlighted the importance of a variety of cardiovascular screening tests per patient type as various characteristics can influence the diagnosis. Moreover, he stated how even though technicians use screening methods such as a cardiac CT scan and an angiogram, these techniques are limited due to the costs and the severity of the condition they test. All of these factors impact the accuracy of the reading and the patient’s outlook.
Dr. Wissam Jaber
Dr. Wissam Jaber is a cardiologist specializing in CAD at Emory Hospital who explained how less invasive early screening devices and the use of a specific biomarker are the current trends in diagnosing CAD, validating the biosensor approach of our project. He also reasoned that even though there are CT scans, angiograms, family history checks, and more, the confounding symptoms present in each patient contribute to a weak basis for accurate testing. Like the other cardiologists, Dr. Jaber emphasized that a biosensing mechanism should detect these miRNAs before detecting calcium as its scores present a higher false positive rate than any biomarker in the bloodstream. He focused on the prevention of the disease progression since early detection meant that patients could take medication and make lifestyle modifications. Therefore, he continuously stressed the necessity of a highly sensitive, miRNA biosensing mechanism.
Dr. Alison Ward
Dr. Alison Ward, a cardiac surgeon at Grady Hospital, a facility that treats underserved patients, helped us redesign our project to address patient differences. Every cardiologist we spoke with previously emphasized the importance of customizable screening tools and tests based on the patient; her daily experiences provided insight into reshaping our perspective on target audiences. She explained that female patients generally have less reliable diagnostic test results due to confounding symptoms such as epigastric pain. Additionally, she mentioned that highly sensitive devices testing troponin are currently being used for diagnosis at Grady, but any device that does not create additional side effects/symptoms, like ours, will be efficient at a healthcare facility. Much like Dr. Jaber, she supported the prospect of patients receiving early medications so that they can make immediate changes to their lifestyle to prevent CAD. Moreover, she advised us to create a more digestible version of the test results since many of her patients do not understand the purpose or implications of diagnostic tests.
Researchers
As we spoke to cardiologists, nearly all of them expressed concerns regarding the feasibility of using miRNA biomarkers in a clinical setting. As a result, we contacted several researchers who are experts in CAD-related miRNA biomarkers to learn more about the ranges at which microRNAs exist in patients with and without CAD.
Dr. Christian Delles
Dr. Christian Delles is a cardiac microRNA researcher at the University of Glasgow. We originally discovered his work from his paper, Systematic Review of microRNA Biomarkers in Acute Coronary Syndrome and Stable Coronary Artery Disease, so we contacted him in early March. At this point in our project, we still had concerns about which miRNAs to select as a biomarker for CAD since different sources contradicted each other on which miRNAs were the most optimal. Fortunately, Dr. Delles provided us with insight, and more specifically, helped us decide on detecting miRNA-1-3p and miRNA-133a-3p, both of which are related to myocytes and thus control the heart muscle. He reasoned that these two miRNAs are consistently upregulated, directly involved in the pathophysiology of CAD, and not closely related to other corresponding diseases such as hypertension, heart failure, diabetes, and cancer. Additionally, Dr. Delles recommended that we choose miRNAs with lower numbers since these were discovered earlier and have been more thoroughly researched, and he established criteria to help us determine appropriate miRNAs for detection for our project CADlock.
As we researched miRNAs, we realized the inconsistencies in the reference ranges of the miRNAs between control patients and those with CAD. Dr. Delles confirmed that disorganized data collection and low sample sizes created these discrepancies and guided us toward creating CADmiR (see Inclusivity), an established miRNA database developed to record reference values for the miRNAs we chose for our 2022 project and other miRNAs related to CAD.
Furthermore, Dr. Delles guided us towards using hsa-miR-150-5p and hsa-miR-122-5p, which were related to the build-up of plaque (instead of the muscular tissues of the heart) during the progression of CAD.
Dr. Robert Meller
Dr. Meller is an RNA research expert from Morehouse Medical Hospital who specializes in miRNA biomarkers and expression. He recommended that detecting miRNAs through saliva is not a viable method due to the limited miR-133a and miR-1 concentrations within it. Therefore, we proposed to detect miRNAs via blood serum as it is the easier method to obtain higher concentrations of our considered miRNAs.
Additionally, we asked about the risk of false positives or negatives, a concern we had heard from many cardiologists due to their increased presence with current diagnostic testing. Because of the limited research available regarding ranges of miRNA biomarkers in patients, Dr. Meller recommended identifying subtle changes in patients by using a concentrated sample size. After reading our findings of up-regulated miRNAs to diagnose CAD, he suggested that we use a control miRNA biomarker (a miRNA biomarker that is downregulated in CAD patients) to compare the results between experimental biomarkers. With this control, there would be a smaller chance of a false positive or negative result. Therefore, we designed this biosensor and submitted it as BBa_K4245209; however, we did not end up implementing this set of experiments this year.
Dr. Charles Searles
Dr. Charles Searles is a cardiologist from Emory University’s School of Medicine who currently researches cardiac miRNA biomarkers. Based on Dr. Meller’s advice to utilize a control microRNA biomarker to compare our other experimental biomarkers, we asked Dr. Searles if he was aware of any miRNAs that could fit this purpose. He recommended using hsa-miR-451a, which is an erythrocyte-specific miRNA with functions related to hemolysis and erythropoiesis, meaning the pathophysiology of CAD is not related to this miRNA (Mussbacher et al., 2020).
After presenting our project to him, he also suggested biomarkers that are correlated with atherosclerosis (a common occurence in patients with coronary artery disease), including hsa-miR-122-5p and hsa-miR-150-5p, which are dysregulated, as well as hsa-miR-126-3p, which is downregulated. These biomarkers may be more useful in use for early screening since atherosclerosis can be tracked over time. The development of biosensors for these miRNAs are part of the future goals for Lambert iGEM.
In addition, we proposed the idea of CADmiR, a specialized database where researchers can add and retrieve information regarding miRNAs related to CAD (see Inclusivity). Dr. Searles supported this idea, as no databases are currently available due to the relative novelty of this area of study.
Broader Community
Organizations
The southeastern United States has a high prevalence of CAD. Lambert iGEM sought out the Georgia Cardiac Office of Health (GCOH) to understand the implications of our project from a government perspective. Not only did the office agree that CADlock would be a valuable diagnostic tool (especially for those in low-income areas that cannot afford screening tests), but they also provided valuable advice on ways to increase the credibility of our tool via data collection. In partnership with the GCOH, we curated an educational pamphlet and survey to be displayed in government health facilities and offices to increase awareness surrounding coronary artery disease (CAD) and learn about our efforts. Lastly, we consulted with the GCOH regarding the legal proceedings a new diagnostic tool needed to undergo; consequently, we created CADlock with the Food and Drug Administration(FDA) approval process in mind.
Furthermore, the GCOH directed us to the American Heart Association (AHA) to learn more about CAD from the patients’ perspectives. The first representative we spoke with gave us insight into the sensitivities CAD patients face, such as diet and medication, which increased our mindfulness when conducting patient interviews. We also met with Going Red, a branch of AHA targeted toward supporting women with CAD, who confirmed our team’s initial conclusions about the lack of inclusivity in the existing CAD diagnostic processes.
Cookbook
Since we identified patient health to be the most impacted by our project, our initial goals were to prioritize patients’ health and experiences. Even though we are not releasing the testing kits to the public, we sought to understand the perspective of researchers and healthcare providers to learn more about the patients before holding conversations with them. Therefore, we reached out to cardiologists and consulted with leading researchers in cardiovascular disease’s correlation with miRNAs.
While validating the benefits of the applications of CADlock in various healthcare facilities, we found compelling evidence in pre-existing studies covering the disproportionate influence of CAD on some ethnic minorities. For example, a survey by Mochari-Greenberger and Mosca concluded that Hispanics and American Indians are often disproportionately affected by CAD (2015). Additionally, Cleveland Clinic emphasizes the same aforementioned groups, along with African Americans and Asian Americans, as of higher risk in cardiovascular disease and precursors for coronary artery disease [i.e of all minority groups, African Americans are cited to have the highest prevalence of hypertension, a precursor to CAD at 59% (2022)]. To address this issue, we created an inclusive cookbook dedicated to disseminating recipes that are not only heart-healthy but maintain cultural integrity.
Recipe Compilation
To ensure that a variety of cultures are sufficiently represented in our cookbook, we relied on a two-pronged strategy to gather recipes:
- Localized recipes from our families’ cultural/ethnic backgrounds and our school’s nutrition department
- Collaborations with iGEM Teams around the world
Local Sources. Our school consists of students from various cultural backgrounds, with a 57.6% minority enrollment rate (see Fig. 10). In collaboration with our nutrition department, we collected homemade recipes and collaborated with our school’s nutrition teacher, Mrs. Shirley Azbell, who provided heart-healthy recipes from underrepresented areas and verified the nutritional information of cookbook recipes (see Fig. 11).
Figure 10. Graph of Lambert High School’s Demographics displaying a 57.6% minority enrollment rate (Georgia
Department of Education, 2022).
Figure 11. Selection of recipes from our nutrition department (Piper Allsup, a student from Mrs. Azbell’s nutrition
class).
Global Sources. Our team collaborated with several iGEM teams around the world including USP.eel, REC-Chennai, and Waterloo-iGEM to expand our collection of recipes, and aid in our goal to find localized recipes (se Fig. 12).
Cookbook Creation
After compiling a collection of heart-healthy inclusive foods, we created a global heart-healthy cookbook with our mascot Chef Threeheart.
Chapter Layout. To better organize our recipes, our cookbook is divided into 9 chapters, each representing a global region used to organize our recipes. The global regions are listed below:
- East Asia
- Southeast Asia
- Middle East
- South Asia
- Sub-Saharan Africa
- Europe
- Latin America
- North America (US/Canada)
- Oceania/Pacific Islands
Each recipe page includes the following:
- Background: Description of the Cultural Origin of the recipe and its characteristics.
- Nutrition: Includes Calories, Sodium, Sat. Fat, Protein, Cholesterol and Carbohydrates.
- Ingredients: Includes a list of ingredients and any extra materials apart from standard cooking equipment.
- Country of Origin: Provides the country which the recipe originated from.
- Instructions: Provides a set of concise instructions, with any preparation steps included beforehand.
- Diet Restrictions: Color key mentioning any dietary restrictions this recipe may have .
View our Cookbook!
Food Bank
To reach prospective patients and address the correlation between socioeconomic status and the risk of CAD, we implemented a food bank initiative to reach out to communities in these impoverished conditions (Clark et al., 2009). This disparity is often associated with the lack of proactive care or confusion on ways to implement a healthier lifestyle. Since a balanced diet and continuous exercise are correlated with a decreased risk of heart disease by about 50%, we decided to implement dietary habits in our initiative to aid underprivileged members of our community (Yeh, 2019). We first researched methods to effectively spread the importance of heart health in an accessible manner and then contacted local food banks to integrate our recipes into our district. The Place, a food bank located in our home county, was extremely accommodating in arranging meeting dates during our correspondence, and we kept in constant contact through emails and scheduled meetings. From our correspondence, we sent a condensed pamphlet full of heart-healthy recipes along with the customizable meal kit from The Place (see Fig. 13).
We took recipes from our collaborations with Waterloo iGEM, REC Chennai, and USP.eel and compiled them into the pamphlet using ingredients provided by the meal kits to expand our outreach. Additionally, we included suggestions on how to implement a healthier lifestyle and a short excerpt introducing synthetic biology and scientific information. We decided to include these scientifically focused excerpts in order to hopefully garner interest and inspire recipients to investigate the synthetic biology field, along with justifying why the recipes were adapted to focus on a heart-healthy approach.