Overview

Product design has been at the front of our project since we came up with our initial ideas. We wanted to spend our summer creating a solution to a real world problem that affects a large proportion of the world’s population. In creating a realistic therapeutic product we have looked into current treatments, safety, manufacture, accessibility and the possibilities for the project after iGEM, throughout each stage we have had the patient at the forefront of our minds. After a range of ideas, we have settled on a final design for a new therapeutic to treat IBD.

Which Autoimmune Diseases?

We sent out a survey for patients with autoimmune diseases in order get a better idea of what patients would like from a treatment. We received 48 responses from members of the public suffering from autoimmune diseases of ages from 18 up to 70. The responses gave us a greater understanding of the current treatments, the problems with them and what patients would like from a treatment. We were astounded by the positivity towards GE and for our project. There is a great deal of belief from the public in the potential of GE in medicine.

They survey told us the public wanted a treatment that treats “a condition at its source rather than masking symptoms.” that was also “less invasive and less dangerous”. They also showed support of a device that was able to provide personalised doses according to the patient. This led to the initial design of our treatment.

We realised that our initial idea of a general treatment was over ambitious. Many of the responses said that they would feel more comfortable if the treatment was designed with a greater focus on select conditions.

We have specifically designed our treatment with Crohn’s disease and Ulcerative Colitis in mind, however, we also found how closely related other autoimmune diseases are and decided to look into which conditions we have the potential of treating if we were to develop our treatment further. Our treatment has a huge range of applications. For our initial market, we would focus on IBD but the range of diseases affected by the intestinal immune system allows our product to be modified for trials on other relevant autoimmune conditions.

Autoimmune enteropathy, Coeliac disease, Rheumatoid Arthritis, Multiple sclerosis, Psoriasis and Type 1 diabetes have all been shown to be linked to the balance between intestinal Th17 and Treg populations (or IL10 concentration) in the gut. For Coeliac disease, we may also need to combine our system with a mechanism to digest or modify gluten. Other conditions, such as Microscopic colitis, Lupus and Hashimoto’s thyroiditis, also relate to at least one of these populations but more research will be needed to find how they are connected to the gut. As well as this, specific probiotics have been shown to be beneficial in a range of these conditions. Our device may act as an appropriate treatment with some minor modifications for these conditions. An imbalance in the immune cells in the gut has been shown to cause an autoimmune condition in the kidneys due to the migration of Th17 cells showing the importance of bacterial population control in the gut.

Types of epilepsy have also been found to relate autoimmunity in the gut. The gut-brain axis may connect autoimmunity in the gut to nervous system diseases and promote Th17 cells in the CNS, similar to what has been found with multiple sclerosis. 80% of cases of epilepsy are in the developing world, this is thought to be due to the influence of bacteria, such as segmented filamentous bacteria (SFB), from water sources influencing the gut microbiome and immune cells. The recent and sharp increase in cases shows the necessity of a simple and effective treatment. If the condition is confirmed to be linked to autoimmunity in the gut then our product can offer an easily administered treatment that stops the disease at its source.

We summarised this data in a table illustrating which autoimmune diseases have been found to be linked to immune cell balance in the gut. References can be found at the bottom of the page.

Current Treatments

To ensure the necessity of a novel treatment we looked into what is currently available and the efficacy and side effects of current therapies. We had already learnt a lot about treatments out of our survey for autoimmune patients. The survey told us that the majority of patients use a combination of treatments and highlighted the problems with their treatments.

We have created a table to summarise current treatments and their associated side effects:

Current Treatment Summary Table

Treatment	Delivery	Action	Success	Side Effects
Aminosalicylates (5-ASAS)	Oral/suppository/enema	Reduces Inflammation	Works for 7/10 mild cases. Ineffective for severe IBD and maintaining remission	Rarely Serious
Immunosuppressants	Tablet	Reduces the activity of the immune system	Effective after 2-3 months	Vulnerable to infection, low red blood cell production
Corticosteroids	Oral/suppository/enema/IV	Reduces Inflammation		Short term: acne, weight gain, mood changes, insomnia. Long term: osteoporosis, cataracts
Ciclosporin	Tablet/7 day infusion	Strong immunosuppressant		Short term: tremor, hair growth, swollen gum, feeling/being sick, diarrhoea. Long term: High blood pressure, reduced kidney/liver function
Biologics	Infusion/injection	Block receptors for inflammation stimulating proteins		Increased risk of infection, vertigo, dizziness, allergy-like reaction
Surgery	-	Colectomy/ileostomy	Relieve symptoms but they usually come back	Patient spends a week in hospital and a few months recovering

Current treatments show no effect for 1 out of 5 patients with IBD and those that do often have very serious side effects. From our initial survey, we found a number of patients who are currently not on any treatment as nothing has worked or the side effects have been too severe and more stated their current treatments are not working⁶. Others were concerned of long term side effects such as increased risk of developing certain cancers. We believe that a new treatment must be developed that is both more effective and more patient friendly.

Professor Simon Travis

We got in contact with Simon Travis, Professor of Clinical Gastroenterology, who has been the President of the European Crohn’s and Colitis Organisation (ECCO) and an elected Member of the International Organisation of Inflammatory Bowel Disease, to gain more of an insight into the current problems with treatments and what a doctor would like out of a new treatment. He believes probiotics have potential in treating Crohn’s but encouraged us to look for evidence showing it’s success. He also thinks patients won’t mind using genetically modified products, they only care that the treatment will improve symptoms.

In terms of safety he believed, especially compared to current treatments such as anti-TNFa, proteins produced by probiotics are far safer as they will be quickly degraded. His concern came from consideration of the environment. He directed our consideration towards the impact the bacteria would have coming out as waste and possible effects in sewage. This is especially important in developing world.

Finally, he encouraged us to think of the strains we would use in terms of its adhesion properties in the intestinal tract rather than just ease of modification. The localisation of IBD is important to consider when choosing a bacteria as we want our treatment to be as close to the source as possible in order to have the greatest effect as possible.

After our meeting with Simon Travis we gained confidence in the use of probiotics and were directed towards further avenues of research in order to ensure our treatment was as efficient as possible.

Combined Treatment

Intestinal epithelial damage is characteristic of IBD due to increased apoptosis of epithelial intestinal cells (EICs). Therefore, a common concern for probiotic treatments is the possibility for bacterial movement across the intestinal epithelium. IL-10, in conjunction with glucocorticoids (a commonly administered treatment for IBD), has been shown to decrease the permeability of damaged intestinal epithelium via an unknown mechanism; IL-10 deficiency has in fact been associated with increased intestinal permeability and development of spontaneous colitis². Therefore, we believe that our therapeutic will have favourable interactions with current treatments for IBD.

The survival of L.lactis is negatively influenced by gastric acid. Previous studies have countered this by co-treating patients with a proton pump inhibitor with the probiotic. For our product we may recommend taking the product with a proton pump inhibitor as well to promote colonisation in the gut

Gene Therapy Case Studies

There are currently very few treatments that are relevant to our project. Gene therapies are on the rise but the cost of these make them unattainable for patients and mean that they can’t be profitable for pharmaceutical companies preventing further research and expansion of the treatment. There are currently 504 gene therapies in clinical trials with 34 of these in the most advanced stage of testing. The sudden surge in this new type of therapy has enlightened many unseen problems shrouded by the confidence in the efficiency of the treatment.

CAR T

CAR T is the first gene therapy approved by the FDA but has encountered many problems. Patient’s T cells are transformed to recognise and attack cancer cells. Kymriah by Novartis fights against acute lymphoblastic leukaemia, 3,100 new patients a year suffer from this and 30% of these people don’t respond to previous treatments. The treatment has a response rate of over 80% and requires only one injection. Although this seems like a miraculous treatment curing a patient is not a sustainable business model especially with the huge $475,000 price tag for the cure. The high cost is from T cell collection, manufacture involving 151 stages with 54 decision points, transport, testing, low number of patients, one administration cure and trained specialists present in only 32 sites in the USA. The requirement of specialists not only increases costs but restricts the locations that it can be administered.

Although the high cost is thought to be cheaper than the lifelong alternative, the one off payment is unaffordable for most customers. This has required Novartis to seek a new business model. If the patient shows no response within a month there is no charge, this increases the customer's confidence yet doesn’t lower the cost. The cost has already been reduced by the FDA permitting smaller groups for clinical trials, even though side effects such as cytokine release syndrome were found resulting in deaths during the trials it still passed. This shows how necessary a new treatment is and how closely managed the administration must be. Novartis is currently looking into reducing costs by the use of donor T cells rather than the patients and by automating manufacturing systems reducing the need for skilled operators.

Genetically Engineered Probiotics

Probiotics are an effective, patient friendly treatment. Genetic engineering of the novel therapy opens up new avenues for treatments. By restricting the genetic engineering to the probiotic rather than the patient dramatically reduces any risks for the patient and allows the treatment to be stopped and the probiotic removed at any point.

GE probiotics are a very new concept, this is demonstrated by how few companies are currently in this market, because of this we have used 4D pharma plc and MaaT pharma as case studies in production and marketing probiotics. 4D pharma plc and MaaT pharma produce natural probiotics, test which strains are effective for which conditions, and research what can be considered a healthy microbiome. Their website contains some basic information about manufacture, clinical trials, finance, patents and more. We used this information with further research to develop our initial model for the manufacture of our product and for our business model.

Whilst at New Scientist Live as well as conducting our outreach work we decided to conduct a survey to get a greater understanding of the public’s support of GE probiotics compared to other gene therapies. Our survey was filled in by school and university students with studying sciences and adults with an interest in science with a range of qualifications. We asked if they agreed with germline cell gene therapy, somatic cell genetic gene therapy and genetically modified probiotics.

The general consensus was that all forms of genetic modification have the potential for good and should be encouraged for the treatment of serious diseases. There was a lot of concern over regulation leading to fear of use for in areas other than therapeutics. The survey illustrated that the public has more support for somatic cell gene therapy and GE probiotics compared to germline cell gene therapy.

Strains

In the lab we have been modifying E.coli, we initially thought we could use this as the probiotic chassis as well as for our lab work. However, as we conducted more research we realised other bacteria may be superior for the treatment of IBD

Dr Chris Barnes

We met with Dr Chris Barnes from the department of Cell and Environmental Biology at UCl to ask for advice for bacterial population control. He directed us to recent studies using L.lactis and agreed they would be safe to ingest and would successfully colonise the gut. He suggested using quorum sensing with a molecule not normally found in the gut to allow accurate control. He also highlighted the variation in the microbiome of different patients would result in large variations in the population of our probiotic. We investigated the range in the bacterial population that our device would be successful at through modelling as you can read about here on our Time Domain Analysis page.

Lactococcus lactis

When we finish testing our device with E.coli we will use Lactococcus lactis as our probiotic. E.coli is safe and easy to engineer but show poor long term colonization of the gut, we have looked into alternatives that would provide a longer term treatment and so require fewer doses. L.lactis is recognised as a safe species and has been engineered greatly including to act as a probiotic producing IL10 for IBD treatment³. L.lactis already has many health benefits so, with our system added, would act as efficient treatment.

L.lactis is able to survive low pH and bile, 98% of the bacteria survive the passage through the stomach and 10-30% survive in the duodenum, which is a relatively high percentage. In the gut the bacteria then adhere to microvilli and form part of the microbiome⁴.

Manufacture

Through studying companies including 4D Pharma plc and Genentech we have developed a rough manufacture pathway.

Manufacture Process

Lyophilisation increases the shelf life of the product to around 18 months⁵. This decreases the requirement for frequent production and batches, and so reducing manufacture and logistical costs. An added benefit of this is for the patient. A longer shelf life will mean fewer hospital visits and a more reliable medication making treatment significantly more convenient for patients. This also gives the option of adding the medication to a yoghurt to encourage colonisation of the bacteria and allows the treatment to be more patient friendly, but it is not necessary and can be taken just as a pill.

Drawbacks

Although we believe our treatment is a significant improvement on what is currently available we must still confront its problems. We have summarised the problems that we have come up with:

• The kill switch means that the antibiotic tertracycline cannot be taken for infections during treatment as this would result in termination of the therapy.


• Activating the kill switch results in production artilysin, this will kill the probiotic but will also spread and kill neighbouring cells. The impact shouldn't be too severe as although artilysin will be present at a high intracellular concentration in the probiotic, it will be a low concentration in the gut when secreted.


• The SoxS/R system is not specific to Nitric Oxide but responds to any oxidative stress. We have countered this lack of specificity by implementing our feedback system which will prevent IL10 production in response to signals, not from T cells.


• The microbiome is a relatively new area of research because of this there are many areas of our treatment that we are unsure about. Interactions with the microbiome and other areas of the immune system have not been fully characterised. However, we can not learn about these further possible interactions until pre-clinical and clinical trials.


• We are unsure about the doses required and the frequency that the therapy must be taken, this will require clinical data.

Final Design

Our aim was to develop a novel medication for IBD with the focus on needs of the patient. Through patient interviews and consultation with specialists, we have learnt of the severity of IBD and the problems of current treatments. These treatments are often ineffective and work by attacking the whole immune system instead of focusing on the cause of the condition leading to significant side effects on the immune system. We have developed a treatment as a probiotic that works on the cause of the condition and so should have fewer side effects. We have incorporated a biosafety mechanism to allow quick removal of the probiotic and comfort the customer. The treatment can be used alongside glucocorticoids to prevent entry of the product across the intestinal epithelium whilst it is inflamed. Through research and surveys with the public, we have decided on a manufacturing process to allow the treatment to be taken as a pill or in a yoghurt allowing it to be a far more consumer friendly than current treatments. We have investigated current gene therapy treatments and regulation to determine how a GE probiotic would compare and what we can do to ensure its convenience as well as how it would be classed by EU regulatory agencies allowing us to determine the exact regulatory procedures we would go through as well as how we can enable our product to reach patients as quickly as possible.

References