When we first began exploring our probiotic, we envisioned the product as a statin-alternative therapeutic. In order to explore the development of a new drug from our probiotic, we consulted with Dr. Sophie Clas. Dr. Clas has more than 20 years of experience working in pharmaceutical R&D and founded PharmaSolv Consulting, a company that focuses on formulation and optimization of appropriate drug candidates.

We learned about the regulatory processes and timeline involved with creation and approval of a new therapeutic. We discussed at length the specifics of animal trials, including histopathology, behavioral response, organ response, bioavailability maximization, pH stability, solubility, and overall safety. Finally, we were encouraged to hear that conversion of cholesterol to coprostanol provides an unambiguous clinical endpoint, which may make for a shorter, less costly trial phase compared to trials such as those investigating certain central nervous system conditions with ambiguous endpoints.

Dr. Clas provided our team with important questions to keep in mind as we develop our probiotic with the end goal of creating a therapeutic drug. The following questions guided the beginning of our research:

• Is the mechanism of the probiotic valid?
• Does the probiotic remain viable in the gut?
• Can the probiotic be formulated into a desired final format?

In order to explore the world of entrepreneurship in biopharmaceuticals, we met with Dr. Cameron Black, Executive Vice President, Discovery, at Repare Therapeutics, a clinical-stage precision oncology company focused on identification of novel synthetic lethality targets and development of inhibitors for these targets.

Dr. Black provided insight on the duration and cost of clinical trials behind drug testing, emphasizing that funding clinical trials requires results from achievable milestones, as drug testing presents a sustained and large investment for venture capital firms. In order to gain funding, therefore, we must generate enough relevant data at each stage to fund the next round of testing and trials, and we must decide how each phase best represents the potential of the drug.

Dr. Black cautioned us to place a great focus on safety, especially in relation to how long a probiotic would remain in the gut and how it would interact with the complex gut microbiome. This inspired us to consider our choice of chassis for our probiotic, specifically in terms of colonizing or non-colonizing nature.

In order to do so, we reached out to Dr. Zack Abbott of Zbiotics to explore their choice of chassis in their engineered probiotic.

We met with Dr. Zack Abbott, founder and CEO of Zbiotics and creator of the world's first genetically engineered probiotic. Zbiotics is a pre-alcohol probiotic drink that mimics liver function and produces enzymes to digest acetaldehyde, an alcohol byproduct that is the primary cause of hangovers.

When we began our project, we had initially planned to use Lactobacillus acidophilus, a gut-colonizing bacteria, as our chassis. Dr. Abbott suggested that it isn't wise to try to "change" someone's gut microbiome, since each individual reacts differently and the gut microbiome is too complex to fully predict the consequences of a modification. He also suggests that from an efficacy standpoint, each person's response to a change in microbiome will be different, so the probiotic should not rely upon a specific gut microbiome response in order to work properly.

Zbiotics uses a bacteria called Bacillus subtilis as the chassis for their hangover cure, which does not colonize the gut and instead passes through. B. subtilis is the same bacteria found in kombucha and tofu, but Zbiotics has engineered B. subtilis to break down acetaldehyde. Based on Dr. Abbott's advice, we adopted B. subtilis as the chassis of choice for our probiotic, providing a consistent effect across consumers due to its lack of interaction with the gut microbiome.

Additionally, the current probiotic market, according to Abbott, is full of faulty science and misinformation. For example, when asking a group of probiotic-takers what they thought probiotics did, nearly all of them answered ”better gut health,” and many were not aware that probiotics were made of live bacteria.

Dr. Abbott believes that the future of probiotics is engineered, and he emphasized the need for better public education regarding both probiotics and GMOs. As such, we partnered with ZBiotics, and are currently producing a video with them on this topic to engage the public and improve sentiment of genetic modification.

Still presenting our project as a statin-alternative therapeutic, we reached out to Dr. Jacques Genest, a leading physician in the Division of Cardiology at the McGill University Health Centre and former Head of Cardiology at McGill University. His primary area of research focuses on genetic bases of atherosclerosis and coronary artery disease. Our team met with Dr. Genest along with Dr. Isabelle Ruel, the National Coordinator for the Canadian Familial Hypercholesterolemia Registry.

Dr. Genest asserted that the most important variable in our project and how it can be used in the medical community is the effect size, or percent reduction in cholesterol. Statins, on average, decrease LDL by approximately 50% and increase HDL by approximately 15%. Seeing as this robust of a decrease in cholesterol levels is unlikely to be produced by our probiotic, he went on to explain that our product is not meant for high-risk patients, patients with familial lipoprotein disorders such as familial hypercholesterolemia, or patients with progressed atherosclerotic cardiovascular disease requiring a sharp reduction in cholesterol levels.

Therefore, from this discussion, it was concluded that our probiotic should be thought of not as a statin-alternative but rather as a preventative for young, health-conscious people who wish to avert high cholesterol before it reaches a progressed stage requiring treatment with statins. This was a major shift in the trajectory of our project, and influenced our entry into the iGEM Food and Nutrition category.

Dr. Genest stated that cholesterol is something that should be managed early on, and the earlier someone is able to control it, the higher the likelihood for a positive outcome.

Our team met with Julia Zumpano, a nutritionist with 18 years of experience in Preventive Cardiology & Rehabilitation at the Cleveland Clinic to learn more about cardiology nutrition and to discuss the challenges that patients face in lowering their cholesterol naturally without medical intervention such as prescription of statins.

The majority of patients that Zumpano sees on a day-to-day basis are those who already have progressed high cholesterol, sometimes due to a familial lipoprotein disorder or other genetic factors, but most often due to poor diet and lifestyle choices. More often than not, patients are not able to lower their cholesterol to the necessary level whether it be due to financial barriers, lack of access to heart-healthy food, lack of time, history of eating disorders, or lack of personal emphasis on heart-healthy eating and regular exercise.

Regardless, a combination of these factors mean that the majority of patients that Zumpano sees are already on statins or are on the brink of needing them. For the former, many patients experience severe muscle aches even with low-dose statins, and for the latter, many patients want to avoid starting statins for a variety of reasons. Zumpano suggests that our probiotic could be used in combination with statins, allowing patients to lower their dosage and reduce painful side effects, and for those who are in the midrange lower their cholesterol to an acceptable level to avoid statins altogether.

We also discussed the potential impacts of pushing LDL too low; according to Zumpano, first of all, what is "too low" is different for each patient based on things like HDL level, family history, lifestyle, and particle size, and is even a subject of debate among physicians. The general guidelines for adults aim to keep LDL under 100 mg/dl, and for high-risk groups a level under 70 mg/dl may be recommended. However, some physicians feel that this level is too aggressive, and that anything under 130 mg/dl may be acceptable, while others think that a lower guideline would be best. Preventive cardiology diets see approximately 20-25% reduction in LDL levels, and she predicts that a probiotic could produce somewhere near the same impact size.

It is certainly clear that in the general patient population high LDL is a much greater risk than LDL that is too low, but she recommends consulting with a physician and monitoring cholesterol levels when starting a new diet or medication, all of which can now be done from home.

This discussion gave important insight on questions of safety and prompted us to look further into ideal levels of LDL and the potential side effects of LDL pushed too low, as well as how to mitigate this when designing our final product. It also introduced the idea that our probiotic could be used in parallel with statins to reduce dosage and thus help patients avoid painful side effects.

Dr. Jennifer Pluznick is a researcher at Johns Hopkins whose research focuses on olfactory receptors in the kidney and their effect on renal and cardiovascular regulation. We learned about her prior research regarding the gut microbiome and its effect on heart health and presented our project to her in detail.

The greatest takeaway from our discussion was that the gut microbiome is complicated, different in every human, and still a domain that we know very little about, which validated our choice of chassis in that we are not modifying the gut microbiome.