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DopaDoser: The Self-Regulating, L-DOPA-Producing Gut Bacteria

Parkinson’s disease is the second most common neurodegenerative disorder with an estimated 10 million people living with the disease at any one point. It affects patients in all aspects of their life and there is an urgent need to improve treatments to allow patients to regain control of their life.

“I can choose to be governed by the illness or I can choose not to be.” (Julia, Parkinson's sufferer)

L-DOPA is the most commonly-used medicine currently available; it can cross the blood-brain barrier and is subsequently converted into dopamine to counteract the dopamine deficiencies in Parkinson’s. Although this therapeutic approach is effective, it can also negatively impact a patient’s quality of life. A main drawback of the current levodopa therapy is that it is administered in oral tablet form. This method of drug delivery can further exacerbate certain symptoms in that it leads to fluctuations in plasma L-DOPA levels, which often causes further motor complications further down the line.

Patients who cannot take oral L-DOPA due to several conditions (intolerance or too high tolerance to oral L-DOPA) are treated alternatively with a so called Duodopa ® therapy. Duodopa 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. Some major problems of this approach are costs of $100,000 per patient per year as well as wear out of the tube every 2 years.

Fig 1 Administration of L-DOPA via bacteria in the patient's gut

Our project aims to tackle problems with current Parkinson’s treatments by introducing a novel, self-regulating delivery system for L-DOPA: DopaDoser is based on the probiotic strain E. coli Nissle 1917 and expresses enzymes necessary for the synthesis of L-DOPA. The amount of L-DOPA produced is regulated via a sophisticated quorum sensing-based system to guarantee that therapeutic levels are reached without leading to peaks and troughs in plasma levels.

In the future DopaDoser could significantly improve patients’ access to treatment and provide a cheaper alternative to Duodopa. The production of L-DOPA via genetically-engineered bacteria would also lead to mitigation of toxic by-products of chemical synthesis and the environmental concerns associated with this. The quorum-sensing system used in DopaDoser could also be applied in treatments for various other diseases as well as improving several industrial processes.

Fig 2 dopamine and L-DOPA biosynthesis pathways

The Graz section is using two quorum sensing based mechanisms for an auto-regulated and time shifted consecutive protein expression, first demonstrated using fluorescent protein synthesis (Fig.3). The fluorescent protein could then be exchanged with genes for the L- DOPA production such that at low cell density levels tyrosine synthesis will be channeled. After a certain biomass is reached L-DOPA production will be produced in a uniformly continuous manner. Alongside the use of fluorescent proteins for the characterization of our regulatory system, we also try to further characterize and improve already existing reporter genes of the biobrick registry. BBa_K1670003 is an improved variant of BBa_K1362461, where two silent mutations were introduced to delete two HindIII sites and a new ribosome binding site as well as a new promoter were added. BBa_K1670001 is an improved version of BBa_E0020, where also a new ribosome binding site and an EsaR/I regulated promoter was added.

Fig 3 Fluorescent protein synthesis dependent on different levels of cell density

Looking to the future our system could be further utilized to activate suicide genes in E.coli to avoid possible overgrowth of the native intestinal flora, which we aim to show proof of concept in both strains common to academic research as well as probiotics, specifically BL21 and Nissle 1917 respectively. Even though we cannot regulate the proliferation of our engineered strain yet, it allows us to provide an outlook of a possible application as a self-regulating drug dispensing system in the GI tract, which may have clinical applications in the future.

Throughout the course of the DopaDoser project we also aim to shape our actions in accordance to the opinions of academics, charities, industry leaders and the public. This includes what implications DopaDoser will have for patients both economically and in terms of quality of life and whether the real world implementation of this technology is feasible. We will also compare ethical opinions throughout outreach about our project and synthetic biology in general across the two countries our team spans and assess this in a wider sociological context. We feel as a team that the issues and human practices surrounding our project are just as important as the project itself and as such we endeavour to tackle a wide selection of concerns.