Team:Oxford/Integrated Practices
Project Selection
One of the main benefits of taking part in iGEM rather than, for example, a traditional summer lab project, is the ability to investigate your project outside of the lab and examine its place in the wider world. Scientific projects do not just exist in the context of the lab, they have the potential to have a huge and reaching impact of society, and so the integration of human practices into these projects is not just desirable, but necessary.
We have integrated human practices research at every stage of our project, from inception to execution, to ensure that we address all the desires and concerns of patients and the public.
This began when we were still proposing ideas for our project, and we talked to the public to determine what kind of issues they would like to be addressed by an interdisciplinary science project. We found that the majority of individuals wanted us to address medical issues. This led us to hone our potential ideas to those that fell under the banner of therapeutics.
Once deciding to address a medical problem, we were very aware that the integration of human practices would be key to our project. Also, we were aware that we would need to address human practices in two main ways:
- Establishing a dialogue with patients and doctors to integrate their requirements and desires into our design.
- Approaching the general public to promote the advantages of synthetic biology and address their concerns with genetically engineered bacterial therapeutics.
Most important to the development of our project was the continuous discourse we maintained with patients and doctors.
Our discourse began when we met with Dr Garry Brown, a medical lecturer who gave the lecture that initially inspired our desire to create a treatment for Wilson’s Disease. Having previously treated patients with Wilson’s Disease, he made it clear that current treatments were unsatisfactory due to significant side effects and had not been improved since the 1980s. He expressed a deal of interest in our idea, and following this meeting we began to investigate copper chelators that could be constitutively expressed by our bacteria.
Patient Feedback
Our initial idea was to produce bacteria that could be ingested prior to a meal that would constitutively express copper chelators before being excreted, taking the bound copper with them. This changed when we talked to patients. Our initial patient contact was Valerie Wheater, who is also the Treasurer of the Wilson’s Disease Support Group. When we spoke to Valerie she expressed the dissatisfaction patients had for the current drugs used to treat Wilson’s Disease: penicillamine and trientine dihydrochloride. She emphasised the restrictive requirement of the treatments needing constant refrigeration, whilst also having to be taken up to four times a day. She also made it clear that many patients were keen for a longer treatment. Even from this initial discussion, we knew that we would have to alter our design to act in the long term, rather than requiring multiple doses throughout the day.
Following this, we spoke to a variety of patients at the Wilson’s Disease Support Group AGM. From these discussions, we identified three key limitations with current treatments, and integrated the ways in which we could address them into our design.
Side Effects
Trientine Dihydrochloride and Penicillamine are currently used to treat Wilson’s Disease. Both have severe associated side effects, particularly Penicillamine. Despite this, due to it being the subject of more in-depth research, penicillamine is usually the first treatment prescribed, followed by trientine if adverse effects are observed.
Serious adverse effects such as bone marrow suppression, anorexia, vomiting and diarrhoea are observed in 20-30% of cases(1). Infrequently, there may be cases of nephropathy (kidney disease) and hepatotoxicity (drug induced liver disease). Side effects associated with trientine include nausea, skin rash, severe stomach pains, diarrhoea, and anaemia(2).
Following the discussion of side effects, we investigated the risks associated with probiotic bacteria, further information can be found here. Side effects from probiotics, if any, tend to be mild and digestive. This concern primarily influenced the choice of chassis that we would ultimately want to use for our therapeutic.
From our safety research and discussion with Professor Kevin Foster (see below), we decided that ideally we would use E. coli K-12 Nissle 1917 to express our bacteria. Further information on risks specific to this strain can be found here.
Price
Both Penicillamine and Trientine Dihydrochloride are very expensive. Trientine Dihydrochloride production in the UK is under the monopoly of a single company, and over the last two years there has been a 600% increase in the cost of the drug, despite the relatively inexpensive production costs. At £3,400 per 100 capsules, it is becoming increasingly difficult for the NHS cover the cost of the drug, particularly as the standard dose is four per day. In the USA, the situation is even worse, with both Penicillamine and Trientine Dihydrochloride having a price of over $22,000 per 100 capsules. These price hikes are making it very difficult for patients to get the treatment they need to prevent their condition from regressing.
One of the intrinsic benefits of developing a probiotic treatment is that our therapeutic has the capacity to provide a significantly cheaper treatment as bacterial cell cultures are relatively inexpensive to maintain.
The impact that price had on the development of our genetic circuit is related to the patient’s desire for a longer term treatment, which is discussed below under “High Dosage Frequency”.
High Dosage Frequency
One of the significant issues that patients expressed the current treatment regime was the requirement for medicine to be refrigerated, whilst also being taken four times daily. Obviously, this requirement is limiting, and patients were keen for the development of a longer term treatment.
Initially, our idea was to produce bacteria that would constitutively express copper chelators. Patients would take these in conjunction with a meal, and excess copper would be bound and excreted. However, this would require constant applications of the treatment.
Following our discussion with patients, where they expressed their concerns with current treatments prices and the high dosage frequency, we decided that a longer term treatment would be necessary. We redesigned our genetic circuitry so that chelator expression would be under the control of a copper-sensitive promoter. This has the advantage of conferring safety benefits - the chelator is only expressed when required. In addition, the metabolic load on the bacterial cells is lowered, meaning that they are more likely to successfully and competitively persist in the gut microbiome. For information on the other safety measures we explored, please click here. To see how wet and dry lab informed the development of our circuit designs, please click here.
Gut Survival
Once we had decided that our goal was to produce a long term probiotic treatment, we spoke to a Professor of Evolutionary Biology, Kevin Foster, to discuss how we could increase the likelihood of our bacteria being able to survive in the gut. He raised concerns over our choice of bacterial chassis and advised us to consider the distribution of different bacterial species throughout the small intestine. He also spoke to us about the possibility of using colicins, on which he has carried out significant research, to make our bacteria more competitive. Along with the desire of patients to take a treatment with fewer side effects, this contributed to our decision to propose E. coli K-12 Nissle 1917 as our final chassis. This non-pathogenic strain naturally produces colicins, allowing it to successfully compete with pathogenic strains in the small intestine(3).
Public and Legal Outreach
Having established our goal, we had two concerns: we wanted to see whether it would actually be accepted by the public, and whether current policy would affect the development of probiotic therapeutics for human use. To address this first concern, we returned to patients and the public, asking them whether they would take a probiotic therapeutic if recommended by their doctor. The majority responded that they would be willing to. To address our second concern, we spoke to Jane Kaye, Professor of Health, Law and Policy, and Director of HeLEX (Centre for Health, Law and Emerging Technologies). She explained that due to the lack of expertise relevant to synthetic biology in the government, and the fast rate at which new knowledge is being acquired, current policy is very vague. She also said that, with regards to new treatments, policy is very dependent on public opinion of emerging technologies. Armed with this information, we questioned the public on their concerns related to genetically modified bacteria and why they thought people may oppose their use as a therapeutic. The responses we received from this survey acted as a springboard for our ethics research, which we hope addresses much of the concerns that were raised. In addition, this meeting guided much of our safety research, and inspired our initial promoter design of a logic gate based system, relying on three inputs. Unfortunately, due to the short-term nature of this project, this was overly ambitious and we redesigned our system and decided to investigate variations of copper-sensitive promoters alone.
Having finalised our system design: copper-chelators under the control of a copper-sensitive promoter, we decided to investigate how we might deliver this system to the small intestine of the patient. We firstly carried out a literature review, investigating the different ways in which probiotic bacteria can be delivered. These included the use of food products, in addition to pharmaceutical methods(4). We then surveyed both patients of Wilson’s Disease and the general public over their preferred method of delivery. The response was in favour of a pharmaceutical method, as opposed to a non-pharmaceutical mode of delivery, with people preferring a pill or bead to other options. Building on this response, and also the work done by both of the previous Oxford iGEM teams, we decided to investigate the use of a bead for bacterial delivery. We have collected some promising preliminary data regarding this topic, using alginate beads that have been alternately coated in layers of alginate and chitosan(5), but this work is still in progress.
We have also integrated patient concerns into our outreach activities, as the patients we spoke to expressed a disappointment at the lack of public knowledge of Wilson’s Disease. Lack of awareness of the disease can lead to discrimination, for example, some patients expressed the unwillingness of employers to hire people with the disease. When carrying our outreach and engagement activities, and on social media, we took care to raise awareness of the disease wherever possible. This included talking on a country radio station about synthetic biology and Wilson’s Disease, writing an article for Bang! magazine about the disease and our project, promoting awareness on social media and our blog, raising money for the Wilson’s Disease Support Group through a cake sale, and discussing rare diseases at our summer schools events.
References:
- (1) Grasedyck, K. (1988) ‘D-penicillamine--side effects, pathogenesis and decreasing the risks’,Zeitschrift für Rheumatologie, 47(Supplement 1: 17-9).
- (2) Frequently asked questions (no date) Available at: http://www.trientine.com/frequently-asked-questions/#faq9 (Accessed: 8 April 2016).
- (3) Hancock, V., Dahl, M. and Klemm, P. (2010) ‘Probiotic Escherichia coli strain Nissle 1917 outcompetes intestinal pathogens during biofilm formation’, Journal of Medical Microbiology, 59(4), pp. 392–399. doi: 10.1099/jmm.0.008672-0.
- (4) Govender, M., Choonara, Y.E., Kumar, P., du Toit, L.C., van Vuuren, S. and Pillay, V. (2013) ‘A review of the advancements in Probiotic delivery: Conventional vs. Non-conventional formulations for intestinal flora Supplementation’, AAPS PharmSciTech, 15(1), pp. 29–43. doi: 10.1208/s12249-013-0027-1.
- (5) Cook, M.T., Tzortzis, G., Khutoryanskiy, V.V. and Charalampopoulos, D. (2013) ‘Layer-by-layer coating of alginate matrices with chitosan–alginate for the improved survival and targeted delivery of probiotic bacteria after oral administration’, J. Mater. Chem. B, 1(1), pp. 52–60. doi: 10.1039/c2tb00126h.
