Interview with Prof. Dr. Joachim Reidl
Owing to our interest in microbial behavior of our system in the human gut, we interviewed University Professor Joachim Reidl from the Karl Franzens University, Graz about our project. Professor Reidl is working at the Institute of Molecular Biosciences and his research interest is in the field of Medical Microbiology.
Dr Reidl mentioned that it will be very interesting to observe the differences in terms of regulation of our quorum sensing based system in E. coli Nissle 1917, compared to ordinary lab strains like E. coli BL-21. He also mentioned that it would be interesting to find out to what extent intestinally produced L-DOPA goes into the blood stream and subsequently how much of it reaches the brain. It needs to be determined how much L-DOPA is produced in vivo compared to production in a standard lab reactor experiment.
It would also be especially important to determine the exact amount of bacteria that need to be administered and cultivated in the gut. Medication such as “Levodopa” is usually administrated to patients in doses from 500 mg to 6000 mg a day . In previous work done on L-DOPA metabolic engineering, researchers developed E. coli strains that were able to produce up to 1.51 g/l of L-DOPA in 50 hours in batch reactors . This meant that we would have to improve these results significantly to develop a production strain that can be administrated directly to the gut.
Another interesting aspect is the molecular mechanism of how bacteria would retain the genes coding for enzymes for Dopamine/L-DOPA synthesis. In the intestine, as opposed to in a fermenter where antibiotics can be added to retain recombinant plasmids, selection pressure would not be present. This would mean that the engineered bacteria would eliminate their plasmids. To counteract this problem, gene integration into the bacterial chromosome could be applied. Selection pressure by knocking out essential E. coli genes and inserting them into the recombinant plasmid could also be employed (plasmid addiction). Still, there is no guarantee for the integrated DNA or the plasmid to be kept intact by the bacteria because of possible cut outs or mutations of the foreign DNA.
The main point we were able to take back to the lab from this discussion was that we will most probably not have to worry about our developed strain outcompeting the human gut microbiome. On the contrary, there is a possibility that the strain may have difficulties reaching the required L-DOPA levels; this is a problem we will have to work on in the future.
 Muñoz, A., Hernández-Chávez, G., de Anda, R., Martínez, A., Bolívar, F. and Gosset, G. (2011) Metabolic engineering of Escherichia coli for improving L-3,4-dihydroxyphenylalanine (L-DOPA) synthesis from glucose. Journal of industrial Microbiology and Biotechnology, 38(11), pp.1845-1852.
Interview with Dr Monty Silverdale
Dr. Monty Silverdale is a consultant neurologist and honorary lecturer in Neuroscience at the Institute of Brain Behaviour and Mental Health at The University of Manchester . Dr. Silverdale’s research concerns Parkinson’s Disease (PD) and non-motor symptoms. He shared his valuable experience in treating PD, gave comprehensive answers to our questions and provided us with useful and specific feedback on our project.
Dr Silverdale told us that patients with motor fluctuations have three options: intrajejunal administration of levodopa, surgery (deep brain stimulation) and apomorphine. As DopaDoser works by administering L-DOPA in the gut, we asked him about the current method of delivery, intrajejunal tubes, which use a Levodopa gel, called ‘Duodopa’. There are multiple problems with intrajejunal tubes. Not only are they prohibitively expensive, but they have other complications: ‘they get infected, they block and move out of the jejunum to the stomach… all sorts of stuff happens.’ For this reason, very few patients can use this option as it can cost tens of thousands of pounds for a single treatment. As our project, DopaDoser, is cheap and non-invasive, we wanted to know more about chronic pain and how our treatment method will improve on current options.
There are multiple non-motor symptoms associated with PD, one of which is thought to be chronic pain. There has previously not been much research into pain in PD. However, Dr Monty Silverdale thinks that approximately ‘80% of people with PD have chronic pains. Some of them have barely got any mobility issues but they still get chronic pain…’. He is trying to see if there is a link and to determine what percentage of people with dyskinesia actually have pain. ‘…some patients have bad dystonia and bad pain, but some have bad dystonia and no bad pain. That is why we are doing the study – we are looking at genetic polymorphisms that might predispose to pain…’.
We therefore asked him about DopaDoser and how he thinks a Continuous Dopaminergic Stimulation would affect people suffering from chronic pain, and if it might reduce symptoms. He told us: ‘If you had continuous dopaminergic stimulation… the pain will be less, because there will be less peaks and troughs – off-period pain goes and dyskinetic pain will go – there still be some pain left, but you would reduce it. Optimizing PD control will certainly improve pain.’
Dr Silverdale also commented on controversy of GMO in patients’ guts. He thought that there would be few problems with acceptability, particularly within the current research community.
As a final pointer, he gave us a few suggestions on how we could improve our project in the future. He told us that producing dopamine in the gut is not a viable idea as it induces vomiting in patients. This made us re-assess that area of our project and re-consider the dopamine pathway. He also mentioned using a dopamine receptor to regulate levels of L-DOPA. He said: ‘could that [a sensor] be a dopamine receptor… Could you use that? If it actually works, that would be pretty revolutionary in PD treatment, I would have thought.’
To conclude, it seems that DopaDoser has the potential to both alleviate pain and be a much cheaper, viable option for Continuous Dopaminergic Stimulation in a multitude of patients experiencing non-motor symptoms or dyskinesia. We took Dr Silverdale’s ideas on board and modified our project accordingly, and hope that in the future our project could be developed further to include use of dopaminergic sensors.
This interview showed:
- Current treatment (duodopa) is invasive and not effective
- Non-motor symptoms are often underestimated and not treated
- Peaks and troughs in medication concentration should be minimized as they can cause internalization of dopamine receptors
- DopaDoser can alleviate pain that is induced by fluctuations in levodopa concentration and treat fluctuations in blood and bowel conditions, sleep etc.
- DopaDoser is cheaper, safer and less invasive
- Patients are likely to accept DopaDoser
- DopaDoser should not synthesize dopamine
- Our project may be improved by a dopamine receptor and MAO-B and COMT inhibitors
Interview with Prof. Dr. Christoph Högenauer
In order to find out more about human gut bacteria composition we met Prof. Dr. Christoph Högenauer from the Institute of Gastroenterology and Hepatology at the Medical University of Graz.
We found out that composition of gastrointestinal colonization is not only different from person to person but also depends on geographic location, genetic disposition, possible disease and current eating habits. Quantification of these bacteria strains is quite difficult as some might only be abundant in very low concentrations. For actual quantification, q-PCR is the method of choice. However, Escherichia coli makes up 0,1-1% off all gut bacteria but up to 50% during enteritis .
Our selected probiotic E. coli strain Nissle1917 is currently sold under the trademark Mutaflor ®. If applied to the patient only once it is observed that the strain does not stay in the gut permanently but has a lifetime of only a few days and is not detectable after a week. Regular administration is still required.
Interestingly during our interview, Dr Högenauer also told us that he was also working with Parkinson’s patients, preparing them for a therapy form called Duodopa ® from Abbott . This therapy especially serves patients that cannot take L-DOPA pills for varying reasons (intolerance or too high tolerance to oral L-DOPA). It is a special way for administration of L-DOPA (Levodopa ®) directly into the human jejunum through a PEG-PEJ tube. This approach allows for a continuous and stable dosage of L-DOPA over the whole day. However some major problems of this approach are high costs (around €700 per week plus costs for placement of the tube and aftercare) and wear out of the tube every 2 years. Also the system needs to be rinsed on a daily basis. The total cost adds up to €100,000 per year per patient.
Obviously this is exactly where our project comes into play! Our idea allows for continuous administration of L-DOPA straight into the gastrointestinal tract which does not only get rid of tubes going into your body but also allows for a much smaller concentration of L-DOPA administration. Levodopa is currently applied through Duodopa® in concentrations of 20-200mg per hour, in contrast to previously mentioned pills in the multi gram range. Gastrointestinal production of L-DOPA would not only allow for a better lifestyle for patients but also discharge health insurance funds.
 Wenzel, K., Kögl-Wallner, M., Katzenschlager, R., Högenauer, C., Pirker, W. and Poewe, W. (2014) P-aktuell: Aktuelles zur Pumpentherapie mit intrajejunalem Levodopa-Gel. Newsletter der Österreichischen Parkinson Gesellschaft, 2, pp.1-8
Interview with Prof. Angela Tod
Professor Angela Tod is the Florence Nightingale Foundation Chair of Clinical Nursing Practice Research at the Faculty of Medical and Human Sciences at the University of Manchester. Her research focuses primarily on access to health services as well as health inequalities during which she has worked with Parkinson’s patients and Parkinson’s UK. 
Our main focus during the interview was to assess access to as well as quality of care for Parkinson’s patients and how DopaDoser could potentially lead to an improvement in those aspects. Prof Tod explained that peaks and troughs in the condition as well as due to medication pose a big challenge to patients saying that “The unpredictable nature of it and the fluctuating nature of the condition destroys people’s lives.” It is this feature of the disease that we aim to tackle with DopaDoser with regulated levels of L-DOPA and therefore counteracting peaks and troughs in plasma levels.
A concern about our project raised by Prof Tod was the need for monitoring, which although very much speculative at this stage, would most probably need to happen regularly in the early stages of the treatment to determine whether therapeutic levels of L-DOPA can be reached. Although monitoring would initially be an added strain on the patient, Prof Tod stated that “the fact that you can actually level things out, the benefit of that would make people make a lot of trade offs.” Meaning that although initial monitoring would be a burden for patients, it would benefit them in the long term which Prof Tod believes to be the most important factor.
We learned that conditions such as Parkinson’s often lead to health inequalities meaning that certain groups are more likely to have access to treatment and support based on social determinants. From this we drew the conclusion that DopaDoser would be able to somewhat counteract these issues as it would enable people to access treatment and lead to less issues with compliance which often affect disadvantaged patient groups.
Interview with Dr. Mariella Kögl-Wallner
To get some more information on the medical aspects of Parkinson’s, we visited Dr. Mariella Kögl-Wallner, an expert in therapy of Parkinson’s patients at the Institute of Neurology at the Medical University of Graz. We mainly talked to her about the day-to-day difficulties faced by people with Parkinson’s disease. Since medications against Parkinson’s disease have become well established, patients do not have any reduced life expectancy. Still, the treatment means a lot of change in the every day life of a patient. Levodopa® has to be taken dependent on the status of the disease several times a day at least 30 minutes before a meal and can result in peaks and shortages of L-Dopa. Those “off” times (periods with no symptomatic relief)  and dyskinesia  often cause a more progressive pathology. Compared to the traditional i.e. oral Levodopa® therapy, the application of Duodopa® already has some advantages. With this therapy method, a mostly constant L-Dopa level can be reached in the blood, meals do not have to be considered and no blockers like MAO-B (monoamine oxidase blockers) have to be taken.
Dr. Mariella Kögl-Wallner told us that her patients are usually very keen to take part in the testing of potentially new drugs. Furthermore, she confirmed that none of her patients would reject a therapy that includes genetically engineered bacteria as long as they see the positive effects of the treatment. Not only the patients but also the doctors always try to stay on top of latest research results on Parkinson’s disease, to find new ways on how Parkinson’s might be treated or some day even cured. Regarding diagnostics, Dr. Kögl-Wallner told us that they sequence a few specific genes at genetic loci where mutations in PD patients have been found. Still, there are groups of patients with no mutations in these loci. These patients might have mutations at other sites in the human genome that have not been studied yet.
All in all, there are still many gaps in knowledge when it comes to Parkinson’s disease. A lot of research is done in this particular area. But ever since 2004 when Duodopa® was approved in Europe no other breakthroughs have occurred. Transplantable dopamine neurons produced from stem cells  or genetic therapeutic approaches might one day become the therapy of choice for Parkinson’s patients.
 Bartus, R., Weinberg, M. and Samulski, R. (2014) Parkinson’s Disease Gene Therapy: Success by Design Meets Failure by Efficacy. Nature, 22(3), pp.487–497
 Science Daily (2015) Parkinson's disease. [online] Available at: http://www.sciencedaily.com/news/health_medicine/parkinson's_disease/ [Accessed 31 Aug. 2015].