Overview

As seen on our Human Practices Silver Medal page (link), public perception and feedback have played a large part in the direction of our project and its design. Since its inception, one group have informed the design more than any other: the doctors and patients themselves. Throughout the project we have maintained a discourse between ourselves and patients in order to tailor our therapeutic to their requirements. These discussions have influenced our project through the design of our genetic circuitry and method of delivery.

As we met with patients, they also raised concerns over the lack of awareness of Wilson’s Disease and other rare diseases in the general public. We integrated this into our public engagement activities.

Discourse

Dr Garry Brown

“The therapies used today are the same as those in the 1980s – zinc and copper chelators. The chelators DO work, but with severe side effects, and there has been little interest in research looking new treatment methods since.”

The initial idea for our project came from a lecture by Dr Garry Brown on inborn errors of metabolism. Dr Brown has had previous experience with Wilson’s Disease, having treated sufferers earlier in his career. He informed us of some of the issues he perceived with current treatments, which have not been improved upon since the 1980s due to lack of research into other options.

Following this meeting, we began our research into specific copper-binding proteins that our bacteria could constitutively express to chelate excess copper. See how dry and wet lab research informed our chelator choices here (link).

Valerie Wheater, patient and Treasurer of the Wilson’s Disease Support Group (WDSG)

We then contacted the Wilson’s Disease Support Group (WDSG), hoping to get the opinion of current patients on our idea and ask them about their concerns with current treatments. We first spoke to Valerie Wheater, the group’s treasurer. She expressed her dissatisfaction with current treatments, particularly the high dosage frequency. She also expressed the general desire among patients for a longer term treatment.

Patients at the 6th AGM

Following our introduction to Valerie, we attended the 6th AGM of the WDSG and presented our project idea before talking to patients to get their feedback. They expressed 3 key limitations with current treatments: side effects, price, and the high dosage frequency. We examined these limitations and returned to the drawing board, altering the design of our project to address these disadvantages.

Side Effects

Concern: 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 Dihydrochloride include nausea, skin rash, severe stomach pains, diarrhoea, and anaemia (2).

Impact on design: Following the discussion of side effects, we investigated the risks associated with probiotic bacteria, further information can be found here (link to safety page). 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.

Insert table from Eric here concerning different bacteria.

From this and discussion with Professor Kevin Foster (see below), we decided that ideally we would use E. coli K-12 Nissle 1917 to express our chelation system. Further information on risks specific to this strain can be found here (link to safety page chassis choice).

Price

Concern: 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 2 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 4 per day. The yearly treatment costs for a patient are approximately £50,000, meaning that the NHS are spending millions of pounds per year on treatments. 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.

Impact on design: One of the intrinsic benefits of developing a probiotic therapeutic 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

Concern: One of the significant issues that patients expressed with the current treatment regime was the requirement for medicine to be refrigerated, whilst also being taken 4 times daily. Obviously, this requirement is limiting, and patients were keen for the development of a longer term treatment.

Impact on design: 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, meaning that it is unlikely that an excess of copper will be chelated that could result in copper deficiency. In addition, the metabolic load imposed 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 (link to safety). To see how wet and dry lab informed the development of our circuit designs, please click here (link to appropriate page).

Professor Kevin Foster

Upon deciding to produce a long term probiotic treatment, we spoke to Professor of Evolutionary Biology, Kevin Foster, about how we could ensure that our bacteria persist in the gut. He advised us to consider the distribution of different bacteria in the small intestine when deciding on our chassis. In addition, he advised us to think about colicins when designing our system, in order to make our probiotic more competitive in the gut flora. This contributed to our choice of E. coli K-12 Nissle 1917 as our final chassis - as this strain produces colicins, allowing it to successfully compete with other E. coli strains, including pathogenic strains (3).

Return to patients and the public

Having designed genetic circuitry and decided to produce a probiotic therapeutic, we returned to the public to determine whether they would be willing to take a treatment of this sort.

We found that the majority of individuals would be happy to take a probiotic treatment, if prescribed and recommended by their doctor. Another portion of those surveyed were unsure, hopefully by providing further information, these individuals may be able to be persuaded to embrace a probiotic treatment.

Delivery

Once we had determined our genetic circuitry, we reached out to patients and members of the general public to determine how best to deliver our probiotic. We carried out a survey in conjunction with the iGEM team from Vilnius, Lithuania, to compare results from our two countries.

Insert Julia’s Survey 3 analysis

From the results of our survey, we decided to use a “jelly-like” bead to deliver our bacteria to the intestine. Building upon the work done by the 2014 and 2015 Oxford iGEM teams, we decided to use an alginate bead coated in multiple layers of chitosan. Read about our results here (link).

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). Sonnenborn, U. and Schulze, J. (2009) ‘The non-pathogenic Escherichia coli strain Nissle 1917 – features of a versatile probiotic’, Microbial Ecology in Health and Disease, 21(3-4), pp. 122–158. doi: 10.3109/08910600903444267.