Team:Baltimore BioCrew/Human Practices
HUMAN PRACTICES
Introduction
Microbiome research is a relatively new field. Major research on it was started in 2007 at the NIH in an attempt to improve understanding of the microbial flora involved in human health and disease. This project was known as the Human Microbiome Project, and it focused on characterizing and identifying the bacteria making up human microbial flora. In addition to pioneering research on the microbiome, this project also introduced societal and ethical considerations surrounding microbiome research in the scientific research community. As our project fits into this new frontier of microbiome research, we felt it was imperative for us to gain a better understanding of these issues and considerations.Our project aims to address the issue of depleted microbe populations in the gut after taking antibiotics. We plan to create bacteria that can produce MAFFs (mucus-associated functional factors, which are proteins that help maintain symbiosis of gut bacteria), which will allow for the recolonization of beneficial bacteria after the use of antibiotics.
Expert Consultations
We reached out to numerous professionals to make sure our project was sound, ethically and logically. We talked to:Jonathan Hansen, MD, PhD
- Dr. Jonathan Hansen is an Associate Professor at the University of North Carolina. He has a research program that studies the effects of chronic intestinal inflammation on resident intestinal microbiota. We contacted Dr. Jonathan Hansen because he published a paper in which he successfully cloned B. theta genes in E. Coli. His knowledge of the transfer and expression of the recombinant B. theta gene in E. Coli would be very beneficial to our project.
Drs. Emmanuel Mongodin and David Rasko
- Drs Mongodin and Rasko are faculty at the Institute for Genome Sciences at the University of Maryland School of Medicine. Both have performed extensive research on the microbiome both in humans and in model organisms like mice. Both work on the relationship between the host and pathogens as well as studying the normal bacterial composition of the human body. Their work was important for helping us to understand the normal composition of the microbiome, how much it can be changed, what we know about probiotics, and how to conduct human subjects research.
Dr. Rosamond Rhodes
- Rosamond Rhodes is Professor of Medical Education and Director of Bioethics Education at Mount Sinai School of Medicine. She is also serves as co-editor of the American Philosophical Association Newsletter on Philosophy and Medicine and on the editorial boards of the international journals Cambridge Quarterly of Healthcare Ethics, Theoretical Medicine and Bioethics, Bioethics, and Clinical Ethics. We chose to reach out to her because she has experience in ethics in microbiome research. Her input would be valuable in gaining a deeper understanding of the microbiome as well as help us to approach our project in a more realistic manner.
Dr. Lauren Oldfield
- Dr. Oldfield is an Assistant Professor at the J. Craig Venter Institute in Rockville,MD. She is studying the response of the microbiome to changes in the environment. She is also an expert in synthetic biology and infectious diseases and her knowledge of biosafety and biosecurity will be important as we design how to implement our system as a probiotic.
Societal Impact and How our Project is Good for Society
Our idea has potential to be beneficial to medicine and broader society. It would be beneficial for patients taking antibiotics because it would prevent them from getting infections like C.difficile and/or developing colitis. It would be a better alternative to probiotics like kombucha and kimchi because they would replenish gut bacterial populations more effectively. It could also make physicians' decisions to prescribe antibiotics easier, as they wouldn’t need to worry as much about patients catching subsequent infectious diseases. Initially, this would likely be a prescription drug. However, after enough time to observe its affects, our MAFF supplement could likely become an over the counter alternative to common probiotics.1. We learned from Drs. Rasko and Mongodin that microbiome research has changed how we think about pathogens infecting the gut. They used to think only of the bacterial pathogen but now they have changed their perspective to thinking about the whole microbiome in the host because there were so many different responses to the same dosage of the same pathogen. So now they question the host’s community. The way they think of it now is that the microbiome can be permissive or immune the that pathogen.
2. We learned that the microbiome poses unique problems for doing research. The microbiome is incredibly complex. We don’t yet know exactly what makes a ‘good’ vs. ‘bad’ microbiome. Using stool samples to measure the bacteria present in the gut is a very indirect measure of our gut microbiome. Especially because we have things like regional diversity (small vs. large intestine, colon, etc.) as well as day-to-day and hour-to-hour changes in the microbiome.
3. From Dr. Rhodes, we learned that the microbiome has changed our ideas of ourselves. A person is no longer an “individual entity”, instead we need to think of it as you plus your microbiome. This raises questions like,
"What is the impact of this on the obligation not to cause psychic distress?"
“How did God get these creatures in us?”
“Who are we?”
"What is the impact on the concept of you vs your property?"
and "Where do the microbes come from -- who owns them, even if they come from discarded material? Is anyone owed compensation?"
How does our Approach Compare to Alternative Methods?
1. Our approach or using a probiotic can be safer than microbiome transplants. We learned from Dr. Lauren Oldfield about the dangers of microbiome transplants. Fecal transplants are growing in popularity, but we must take care to not let one bad incident (death from fecal transplant earlier in June/July) affect possibly life-changing therapeutics. Transplants involving stool use the entire microbiome and therefore are sometimes dangerous to transplant.
The main risk involved with our idea was introduced to us by Rosamond Rhodes, a Professor of Medical Education and Director of Bioethics Education at Mount Sinai School of Medicine. We learned from her that the transmission of strains could be dangerous, especially for non-patient populations or especially vulnerable populations (infants/immunocompromised people). Microbiome research should therefore be analogous to systemic infectious disease research. Researchers should consider using infectious disease research protocols such as airflow control.
What we can do, and are doing with this iGEM project, is to isolate specific strains that are responsible for the benefits. Our genetically engineered strain would not have the risk of passing on a dangerous pathogen when patients take the therapy. Right now, we are not good enough to NOT use stool samples for microbiome transplants, but perhaps our approach could be a complementary therapy.
Potential Risks
However, our idea has a few risks. We were introduced to some of these risks through our interviews with bioethicists and microbiome researchers.1. One problem with our idea is that the microbiome of every person is different. There are many factors that affect its makeup, for example diet, genetics, and environment. This is problematic because it means that our idea could have different effects on different people; for some it could replenish lots of gut bacteria and for others it may not work at all. Dr. Rhodes also suggested that a drug that could replenish gut bacteria, like ours, could encourage physicians to write more antibiotic prescriptions. This could have very serious health implications for patients that our probiotic doesn't help; more antibiotics without the help of a probiotic to replenish their gut would make them more susceptible to infectious diseases.
2. Work with the microbiome has also raised privacy concerns. People might worry that microbiome information can be used to identify a person. The particular composition of microbiome varies between individuals, so some people think that you could use the microbiome to track or trace the movement patterns of a person. For example, you could identify the user of a keyboard by looking at skin microbiome left behind. These concerns are for the most part not justified. Drs. Rasko and Mongodin explained to us that the dynamic nature of the microbiome makes it nearly impossible to identify someone; the state of a person's microbiome changes daily. Dr. Rhodes reiterated this sentiment; she told us that there are no examples of harm done to a person caused by the disclosure of research information about them. But, this issue of security of your microbiome that is left behind could be a problem in the future.
3. We need to consider the ethics of Human Subjects Research since we will eventually be giving people a probiotic. Informed consent is needed with human patients. The head of a research team looks at the research protocol and considers the risks, regulation, outcomes, and what knowledge they want to gain. Is the knowledge they are trying to gain worth the risk? The protocols can take a LONG time to get reviewed. Dr. Mongodin had to take a half year to complete it. The IRB (The Institutional Review Board (IRB) is an administrative body established to protect the rights and welfare of human research subjects recruited to participate in research activities conducted under the auspices of the institution. It is very strict and analyzes the risks. We will need to consider who should review the risks to patients of our microbiome therapy.
For the microbiome, the issue extends to informed consent and keeping microbiome samples in biobanks. To inform someone appropriately in a study, you need to include the number of participants, length of study, and expected uses of the data/samples. But these not generally known for samples in biobanks. An article in the American Journal of Bioethics stated that informed consent is not necessary for sample biobanks. Biobanks present a “minimum risk” for patients. There are no examples of harm done to a person by the disclosure of research information about them, and being identified does not constitute harm. Dr. Rhodes suggested that we need to find a balance between de-identifying samples and preserving anonymity and also allowing for future usefulness of the information and samples. For example, separating samples from Electronic Medical Records keeps privacy, but may prevent future uses of the samples.
Integration
1. We used the information that we learned from the experts about the potential safety concerns of genetically engineered organisms by including a kill switch in our design. Our kill switch is part BBa_K3301003
We learned from Dr. Oldfield that the idea of "Dual Use Research" is that scientists are hoping to contribute to the scientific community and fight diseases, but others could use their research to build bio-weapons more easily. Thus, research should be conducted and published responsibly. We also learned that "Dual Use Research of Concern" is another categorization of restricted organisms. For example, viruses that are not commonly found in the United States are in this category. One consideration for combating the use of research in a dangerous way is to split up the information into multiple publications. Fortunately, specific grants and PIs are dedicated to thinking of counter-measures for potentially dangerous genetic mechanisms (for example, against gene drives or CRISPR/Cas9). Our research is unlikely to be used to harm people, but from the beginning of our project, we thought about how we could prevent the spread of these bacteria, since MAFF proteins encourage bacterial growth!
We integrated the idea of a kill-switch into our system early on. Dr. Oldfield suggested that it would be best to have two kill switches! This is not very efficient, but it is always good to have a redundant mechanism. Or, we could make E.coli dependent on an amino acid so the bacteria would die without our taking the amino acids. She also suggested that we have to think about being able to not only contain it in the individual being treated, but from spreading out into the physical structure of the community through waste (sewage). So, we might have two kill switches-one that would prevent it the bacteria from growing too extensively in a person and one to prevent it from growing in the environment.
2. We learned from Dr. Mongodin and Dr. Rasko that they give questionnaires to patients in order to combat the ethical concern of informed consent. A main issue they faced in their research was making sure that subjects knew what what the research that they were a part of entailed. Before performing tests on subjects, Mongodin and Rasko often give lessons and quizzes to subjects to ensure that they were well-informed about the research they are a part of. Despite these efforts, Drs. Mongodin and Rasko told us some patients still had a hard time understanding the research they participate in. This ethical issue was reinforced by Dr. Rosamond Rhodes, who told us that the issue of informed consent is almost as old as scientific research itself: Walter Reed used English and Spanish documents to get consent in 1900s for yellow fever experiments. She additionally introduced us to the Nuremberg Codes, which is a set of rules and concepts about for human experimentation made after World War II. In this document, the importance of informed consent is emphasized; it is the very first code on the list. It states, "The voluntary consent of the human subject is absolutely essential." This idea was reinforced by later documents that outline human research principles, such as the Declaration of Helsinki (1964) and the US Common Rule (1981).
We have decided that we will incorporate the same method that Dr. Rasko and Dr. Mongodin use if we needed to work with the public or patients in the future. Because they told us that patients still have a hard time understanding the ideas of the microbiome and the problems with antibiotics, we responded by engaging in extensive Public Engagement, including a trivia game at Artscape, participation in the Minterlab outreach program, and engaging people with Agar Art. This also gave us the idea to educate the Baltimore community through a flyer (below), which we posted in highly trafficked areas including local stores, schools, and hospitals. This became a large part of our public engagement.
3. When we were researching the transfer and expression of the recombinant B. theta gene in E. Coli, not many references were available, as MAFF was relatively new. Since Dr. Jonathan Hansen published a paper in which he successfully cloned similar B. theta genes in E. Coli, we decided to reach out to him for advice. Dr. Hansen shared with us that many proteins could be toxic to E. Coli growth and suggested that we should only express part of our gene. He advised that we should exclude the signal peptide from our recombinant gene and use an expression vector with tight induction control. Following his advice, we cut out the signal peptide from our designed construct and used the T7 promoter which has very tight control in E. coli cells. Without his help, we probably would not have designed our construct the way it is now and would have spent more time fixing the issue.
4. When we talked with Dr. Rasko and Dr. Mongodin, we learned that the host response to things like probiotics and antibiotics can vary dramatically. They use mice because mice are more uniform in their microbiome. For our long term plans, we changed our direction and now instead of going straight into people for our next step, we think that mice might be the best model organism for the next step of our experiments.