Schistosomiasis is a devastating neglected tropical disease (NTD) that affects over 240,000,000 people globally and results in 200,000 deaths annually. Despite these shocking statistics, the parasitic disease is currently widely unaddressed in many impoverished third-world countries where it is most concentrated. Millions of families around the world continue to live at high risk of infection because of poor water sanitation and limited health care availability.

The standard method to treat schistosomiasis is the antischistosomal drug praziquantel, which while effective in treating infection temporarily, is unable to provide long-term immunity or prevent reinfection. There are also many socioeconomic barriers present that prevent widespread access to such medicines, leaving infected patients untreated and at risk of severe organ failure or even eventual death.

For these reasons, our team, CCA_San_Diego, decided to focus our efforts on minimizing schistosomiasis cases through the natural synthesis and controlled release of sanguinarine—an antischistosomal drug—into environments where schistosomes are prevalent. While this solution may be effective in eradicating parasites out of snail populations, the potential for the release of genetically modified yeast comes with many environmental and ethical concerns. Thus, throughout our project, our team centered our human practices approach around the conversations with our community, as well as in-depth discussions with schistosomiasis experts about the greater implications of our solution.

Introduction

The issue of schistosomiasis does not directly affect the region where our team is based, so we wanted to design an approach to Human Practices that would best incorporate the values of the general public and expert opinions to design a good solution. We knew it would be difficult to travel to heavily affected areas or gain connections with people infected by schistosomiasis, so we depended greatly on broader opinions about the ethics and safety of our solution. Throughout each stage of our project, we engaged in many conversations to best align our project with our main priorities.

Our project used "The Three Triangles Process" (diagram located below), which we chose for the simultaneous convergence of the Discovery and Ideation stages to the initial solution we developed. Through our discussions with schistosomiasis experts, we gained not only a deeper understanding of the problem of schistosomiasis, but also received advice on how to proceed with our project design. This dual action enabled us to constantly refine our solution through a consideration of the broader environmental or ethical impacts and the technical aspects.

Three Main Stages

Discovery This stage was focused on gaining a deeper understanding of schistosomiasis as a disease, as well as the societal issues and health problems that are currently posed by schistosomiasis. We initially selected schistosomiasis as our target disease because of its widespread impact on human livelihood globally but lack of concrete solutions to tackle the issue. To gauge the public perception of NTDs, and specifically schistosomiasis, we conducted a Human Practices survey that asked respondents to rate their awareness of schistosomiasis and the necessity for a novel solution. Our team also reached out to many schistosomiasis experts, such as researchers and physicians, to understand snail-parasite interactions and the onset of disease in infected humans.

Ideation Simultaneous to "Discovery" was the process of developing our initial solution. Our lab team underwent a thorough process of literature reviews to understand current approaches to the schistosomiasis problem, as well as potential avenues for our synthetic biology solution. After settling on the production of a natural compound to directly kill schistosomes, we received input from many professors and researchers about the feasibility of the prototype and the selected compound, which at the time was still plumbagin. We constantly revised our solution to be scientifically sound and feasible.

Delivery After the development of our solution, we began to research how we could implement our bioreactor containing the genetically modified yeast across high-risk areas in an environmentally safe and ethical manner. In order to understand the main concerns of our community about the implementation of our device, we returned to our Human Practices survey to analyze the comfortability of respondents regarding use of genetically modified yeast to treat contaminated water. Our team also conducted a policy review of current African state laws that could affect the implementation of our device, such as synthetic biology regulations and authorities over nationally-owned waterways.

Three Main Milestones

The Brief At the beginning of our iGEM journey, our team brainstormed potential project ideas and were immediately intrigued by antiparasitic compounds produced through genetically modified organisms. While there are many parasitic diseases prevalent today, our team narrowed down on schistosomiasis because it is the second most dangerous disease and found in many underdeveloped nations. With our initial idea in mind, we began to research specific natural products with antiparasitic properties and the method of drug delivery.

The Concept The convergent thinking of "Discovery" and "Ideation" led to the development of our most promising solution to treat schistosomiasis—genetically engineering yeast to produce sanguinarine to kill schistosomes in infected snails.

The Product We developed a cohesive design and approach for implementation. First, we designed a CAD model and physical 3D-printed prototype of our bioreactor that would produce yeast for plates on the shores of infected waters.

Environmental Safety

Schistosomiasis is a waterborne parasitic disease that is often found in tropical areas due to its intermediary host—the freshwater snail. This means that our yeast bioreactor must be located on the shores of freshwater bodies of water to ensure snails uptake the yeast, and consequently, the antiparasitic sanguinarine compound. While this may treat snails of infection and prevent transmission to humans, it also comes with a myriad of environmental issues that must be taken into consideration for safe implementation of our solution.

Despite studies showing that genetically modified yeast do not have increased survivability rates compared to wild-type yeast, it would be irresponsible to release our engineered yeast without any regulations or monitoring, especially considering the controversy that still surrounds synthetic biology in the environment. We needed to ensure our compound was not fatal for the freshwater snails or other native species, which are important ecologically in the aquatic ecosystem.

Ethical Responsibility

Synthetic biology is a novel field with lots of potential in producing solutions for modern problems, but with these advancements come many ethical concerns. We knew that synthetic biology was necessary to achieve the goals of our project and reduce schistosomiasis presence in the environment, so our team used a human-centric approach to engage in conversations with experts and the community.

As we saw through our Human Practices survey, 34.5% of respondents still believe that synthetic biology is not an ethical or safe practice. To ensure we align with these ethical goals, we conducted an extensive selection process for our natural compound to select sanguinarine, as well as an environmental analysis of our solution. Before finalizing the method of delivering our antischistosomal drug, we consulted many synthetic biology professors and schistosomiasis experts to understand the ethics of our solution. We underwent a constant cycle of research and development to ensure that our final solution, delivering sanguinarine on land via yeast, was the most ethical approach.

Scientific Feasibility

We have aspirations for our projects to solve root causes of schistosomiasis, but we also had to be realistic in how much our project could achieve without compromising the health of freshwater ecosystems. We knew that if our project was very far-fetched and unfeasible, implementing it would be very dangerous and could potentially fail. Therefore, we made sure to work with our lab team to find an acceptable balance between potential problems of SchistoGONE and how much we could help the current situation of schistosomiasis.

For instance, one concern brought up to us through our HP survey was that the parasites would eventually develop resistance to sanguinarine. However, we decided that this problem of resistance applies to all drug discovery, and that its advancement should not be hindered altogether. We did research appropriate doses to give to the schistosomes to ensure they are killed without being toxic to its hosts.

Overview

Our team used a digital survey to gather information about the main values, priorities, and concerns of the public. The survey asked questions covering the safety and ethics of synthetic biology more broadly, as well as its application in tackling neglected tropical diseases. We received a total of 336 responses, which came from a diverse pool of respondents. The data we collected was essential in designing a responsible, ethical, and safe solution that aligns with the perspectives of the public.

From this survey, we found out that many people in our community were unfamiliar with NTDs, the basis of our project. Because of this information, we made our Schistosomiasis PSA website to educate more people about a disease that we think needs immediate attention and solutions.

We also found that our community believes that we have a very strong ethical obligation to inform our consumers that our device contains modified organisms. Although most people also responded that they would be comfortable using our product, quite a few people were also skeptical about it, expressing that they were worried about our compounds or yeast escaping into the environment.

We heard these considerations from our respondents, and because so many people were concerned about the disruption of ecosystems from releasing the compound directly and what would become of dead worms in the water, we changed our project's design. After the survey analysis, we were motivated to switch to the snails consuming the yeast directly instead of releasing plumbagin (the compound we were considering then), into infected waters.

Note: Our survey analysis was conducted before we had finalized our project idea. The reference to plumbagin as our compound and bacteria as our host to create the compound is inaccurate to our final project.

Ethical Concerns

From the survey analysis, we also got responses on ethical concerns that the public may have with our project. Respondents had an option to comment on potential problems we may have. Our team responded to each concern and explained how we addressed it. We're very grateful to have a supportive community that helped us to develop and shape our project to be as ethical as possible.

On October 3rd, as a collaboration, our iGEM team, DNHS, and WVHS held an ethics symposium at the J. Craig Venter Institute. Because we saw that many people in our respective Human Practices surveys did not know much about synthetic biology ethics, we wanted to educate the San Diego community of how ethical practices are maintained in research. We had an impressive lineup of iGEM members, student researchers, and professional researchers give presentations about ethical issues in research today, and what ethical values they uphold. It was an amazing opportunity to not only educate our peers and learn from experts, but also to meet each other in person!

Discovery
Dr. Conor Caffrey

Professor Conor Caffrey works in the Skaggs School of Pharmacy and Pharmaceutical Sciences in molecular and biochemical parasitology. He was the first professor we met with and became a crucial mentor to us the entire season.

Takeaways We gained crucial insight on the importance of environmental safety in both the choice of anti-schistosomal compound and implementation of the project. Ultimately, his guidance steered our project in the direction that SchistoGONE currently is in.

Moving Forward Dr. Caffrey allowed us his expertise in Schistosoma Mansoni and his continued guidance through much of the season, which has been integral to our project in terms of environmental safety, antischistosomal effectiveness, and implementation. We are extremely grateful to him and his work at the CDIPD as the CCA team was able to receive data on our antischistosomal compounds tested on live schistosoma sporocysts.
Dr. Joshua Widhalm

To get a better understanding of plumbagin, we met with professor Joshua Widhalm from Purdue University. Dr. Widhalm provided solutions on how to utilize plumbagin without toxicity as an environmental issue.

Takeaways We learned that internal concentration of plumbagin is not the same as the external concentration and that the toxicity level varies. He also informed us that plumbagin can break down in sunlight, so it wouldn't be a major issue. In order to prevent autotoxicity, we needed to transport the plumbagin during synthesis.

Moving Forward Professor Widhalm encouraged us to look closer into plumbagin and methods to prevent toxicity in ecosystems. Although we ended up changing plumbagin to sanguinarine a few months later, Dr. Widhalm's knowledge taught us how to explore methods of making a compound lethal to schistosomes, but not other organisms in the water.
Dr. Sharon Reed

Dr. Sharon Reed is an infectious disease specialist at UCSD Health. She also does research on antiparasitic drugs, which we were interested in learning more about. As a physician with firsthand experience in schistosomiasis, Dr. Reed provided us with detailed information about its diagnosis and symptoms.

Takeaways Reed gave us insight into schistosomiasis as a disease and how it has been treated in the past. For example, we learned that many efforts to treat schistosomiasis in the military have been made, one being a topical medicine after exposure.

Moving Forward Armed with the new perspective and examples of treatment in real life, this aided us greatly in selecting our way of implementation of our schistosomiasis prevention.
Dr. Si-Ming Zhang

Professor Si-Ming Zhang works at the University of New Mexico and researches snail intermediate host and schistosome parasite interactions. In the beginning stages of our research, we were lacking information about the actual snails themselves and their life cycles, so we contacted Dr. Zhang for a meeting.

Takeaways We learned that worms use a chemical gradient to find snails to infect and in which part of the worm's life cycle we should try to kill it. Initially, we were planning on trying to find a way to kill the cercariae form of the worm (when its mature and looking for a human to infect), but he advised us to kill the worm in its miracidia (when the worm is swimming and finding a snail) or the sporocyst (inside the snail) phase. He also helped us gain access to research that we did not have access to.

Moving Forward Using the research and advice that Dr. Zhang provided us, we decided to target the miracidia and sporocyst phases of the worm's life. This really helped us narrow down where we would target the parasite, inside the snail, by focusing on only a few life cycles.
Ideation
Dr. Elizabeth Winzeler

Professor Elizabeth Winzeler works in the division of Pharmacology and Drug Discovery, as well as the department of Pediatrics and translational research at the UCSD Health Sciences Center for Immunity, Infection, and Inflammation. We contacted her because of her expertise in malaria drug discovery for both prevention and treatment.

Takeaways Dr. Winzeler introduced some potential issues with our project, including the costliness. effectiveness, and ethical concerns of our method of dispersing our product. She also brought up that releasing E.coli into the water could pick up plasmids and be lethal, so we may want to look into modifying the snails themselves. She also encouraged us to reach out to communities and governments in Africa and areas where we want to implement SchistoGONE in the future.

Moving Forward When continuing with our project, we made sure to take into consideration the concerns Professor Winzeler had. Because of her concerns with E. coli and plumbagin, we reconsidered these aspects of our project and eventually changed them. Professor Winzeler was integral in troubleshooting potential environmental issues that our project may cause.
Dr. Karin Fester

Dr. Karin Fester works at the University of Leipzig in the Institute of Pharmacy. As a phytochemist, she published a paper on an attempt to produce plumbagin in E. coli; however, it did not work initially because they did not have the full biosynthetic pathway down due to missing enzymes. One of our team members encountered Dr. Fester's paper and found it very relevant and insightful to our current project, so we contacted her to see if she would meet with us.

Takeaways From Dr. Fester, we realized that instead of trying to produce plumbagin in E. coli, which is difficult due to missing enzymes for the biosynthetic pathway, we should use yeast because it has an endoplasmic reticulum and is a eukaryotic expression system. Eventually, we decided to use saccharomyces cerevisiae, a species of yeast that has already been used for much research and showed success. Dr. Fester's advice was also key in our switch away from plumbagin as our compound. She recommended pimaradenoic acid, which is easier to produce.

Moving Forward From our meeting with Dr. Fester, we changed many aspects of our project. We switched away from both E. coli and plumbagin, and although we had to conduct a lot more literature review and research for this change, it ultimately made our project more feasible.
Dr. Winka Le Clec'h

Dr. Winka Le Clec'h is a staff scientist at the Texas Biomedical Research Institute. Because of her work in schistosome drug resistance, she is an expert in snail-schistosome interactions—information that we lacked.

Takeaways From Dr. Le Clec'h, we realized that releasing a compound to feed the snails under lab conditions was possible, but in a realistic setting, would be much more difficult because the plasmids could be easily incorporated. She advised the usage of yeast, as pheromones can attract the snails and that they should be consuming some kind of compound. The most important thing she taught us about was the immune response of the snail once it is invaded by the schistosome. If the snail and schistosome are compatible, the snail will fail to have a successful immune defense. However, if they are incompatible, the snail will be able to kill off the worm.

Moving Forward Dr. Le Clec'h was very important in our project design because she got us thinking about the snails' immune responses to the parasite. We got the idea of possibly exploiting the snail's immune response to kill off the parasites immediately upon their entrance.
Dr. Larissa Podust

Professor Larissa Podust conducts pharmaceutical research at UCSD. As a structural biologist, we were interested in her research of drugs targeting parasitic diseases. At the time, we didn't have much information on how the compound itself would work, and had mainly focused on its production.

Takeaways We focused on the specificity of drug delivery: how would we deliver our compound in the most effective manner? Dr. Podust encouraged us to think about how to maximize the specificity of our drug to ensure that the parasites, our target, would be able to come in contact with sanguinarine and die. We also learned a bit about drug kinetics: how quickly would the drug work and degrade? We realized that the compound would not be effective if it degraded before reaching schistosomes.

Moving Forward We crossed off releasing it through a water-based delivery mechanism on our list, and instead started narrowing down the targeting phases or attracting snails. We ended up combining the two ideas, attracting the snails into eating the substance which then targets sporocysts in the cells.