Team:Nottingham/Poster

Nottingham

NeuroTone: The Microbiome and Neurodegeneration

Abstract

The University of Nottingham iGEM team have been developing a novel biotherapeutic to delay the onset and progression of neurodegenerative diseases, using synthetic biology.
By engineering the bacterium Clostridium sporogenes to secrete the ketone D-β-hydroxybutyrate (DBHB), we achieve neuroprotection by ketone-mediated relief of oxidative stress in the brain. This will occur via delivery of our C. sporogenes spores to the gut, where an established culture will produce DBHB - which enters the blood stream and crosses the blood-brain barrier, utilising the gut-brain axis.
Using mathematical modelling, we identified a pathway to favour microbial DBHB production and have investigated how the culture size and metabolic activity could be regulated. Recognising potential concerns regarding genetically modified organisms, we designed strict controls to ensure our biotherapeutic cannot escape into the environment. Through several outreach projects alongside consultations with key stakeholders, we have engaged the wider community to inform and shape our work.

Meet The Team

James Birch is this year’s student team leader and is also working on the Biocontainment subgroup of the project.

Saachi Bhalla is this year's deputy leader and is part of the DBHB subgroup.

Luke Barks is part of this year's innovative modelling subgroup and is also responsible for marketing and branding.

Aly Sadowska is also one of our innovative modellers as well as being responsible for marketing and branding. Aly has produced many of our amazing illustrations!

Eugenia Vuong is the head of collaborations and is in charge of our wiki page. She is also on the DBHB pathway subgroup.

Kieran Bird is head of outreach, human practices and ethics. He has worked hard making our human practices integral to our project. He is also on the routes of administration subgroup.

Alistair Cadoo is head of communications and fundraising and has kept our social media active and engaging. He also works on the routes of administration subgroup.

Luke Weir is our team recording officer. He has done a lot of integral research for our project and was the creator of the project idea. He also works on the Biocontainment subgroup.

Introduction

The Issue

Neurodegenerative diseases are caused when neurons in the brain are damaged and start to function abnormally. As the cells die, signs of degeneration may start off relatively mild - such as forgetfulness and hand coordination changes.

However, this can quickly spiral into the more severe symptoms of memory loss, anxiety and loss of balance and bodily control.

This has huge debilitating effects on the sufferers – independence in daily life changes dramatically and may even be lost altogether. Neurodegenerative diseases affect all aspects of life, extending to loved ones and the much debated state of the care sector.

It puts pressure on the immediate family to become carers and look after their (often elderly) relatives. Inevitably, the extra stress and worry can become too much and means considering sending them to care homes to be looked after.

We recognised this as an important starting point for our research early on and aimed to include someone working in the care sector to better understand what our project should achieve.

The Stats

Neurological long-term disability has a huge impact on quality of life. In a single year, it was calculated that the number of years of a good quality of life that have been lost to neurodegenerative disease, totals more than 276 million years.(1)

The Solution?

By integrating human practices, we realised that patients and carers needed a new way of administering medicines, particularly for neurodegenerative diseases. They deserved treatment that should be proactive rather than reactive – when it may already be too late.

This year, the University of Nottingham 2020 Team have come up with the project NeuroTone.

- A new way to administer biopharmaceuticals for neurodegenerative diseases using a Clostridium spore-based delivery.

With our growing and ageing global population, it is more important than ever to tackle this current crisis by using the latest in science and synthetic biology innovation.

Inspiration

Neurodegenerative diseases currently affect a large proportion of the population.

Dementia is the leading cause of death in the UK.

As they disproportionately affect the older ages, the future looks dim due to the ageing population in many developing countries, including the UK. Current treatments all focus on alleviating symptoms. This is due to there being no single known cause of these kind of diseases - although multiple causes have been hypothesised. There is clearly a gap that needs to be filled or at least thought about before these diseases become more prevalent.

Figure 1: The four leading causes of death in the UK from 2005 to 2017 (https://www.alzheimers.org.uk/blog/research-dementia-UK-biggest-killer-on-the-rise)

As the intermittent fasting diet becomes more popular, it is interesting to look at its benefits and the scientific basis behind them. One main mechanism of intermittent fasting is the use of fatty acid-derived ketone bodies for energy by many organs including the brain. The bloodstream concentration of ketone bodies such as DBHB also clearly increases during intermittent fasting. Various papers show either the neuroprotective effects of DBHB(2) or the use of intermittent fasting diets in treatment of Alzheimer's disease.(3)

These two factors linked together to inspire our project and lead to an interesting and novel way to think about the treatment of neurodegenerative diseases.

Routes of Administration

Our team identified 4 requirements to keep in mind during the design of NeuroTone. Clostridium sporogenes, our selected strain, satisfied these as shown below.

Figure 1: Diagram highlighting the requirements that shaped the design of NeuroTone, and how those requirements were met.

How would NeuroTone be administered?

NeuroTone would be delivered to the gut as a capsule which would break down through the digestive process – releasing our engineered C. sporogenes.

Figure 2: Representation of the NeuroTone capsule and its components.

What happens when a patient takes NeuroTone?

The NeuroTone capsule is dissolved when it reaches the intestine due to a change in the pH (4) - releasing the spores. C. sporogenes is an anaerobic organism, so when the oxygen levels are low enough the spores germinate and the vegetative cells are then able to synthesise DBHB through the integrated pathway our DBHB team designed. This neuroprotectant enters the bloodstream and is carried to the brain, where it is able to protect the neurons and prevent their degeneration.

Figure 3: Representation of the route our biotherapeutic follows inside the human body. The NeuroTone capsule is administered orally and dissolves in the gut. Then, the C. sporogenes spores germinate and DBHB is produced – the neuroprotectant is transported to the brain through the bloodstream.

DBHB production pathways

Our team set out to discover and engineer synthetic DBHB production pathways for use in our C. sporogenes biotherapeutic.

Constructing DBHB production pathways

• When constructing possible DBHB pathways we concluded the most viable route to producing these ketones using the components of the Acetone-Butanol-Ethanol (ABE) fermentation pathway.
• From within this pathway the DBHB subgroup hypothesised synthetic pathways to produce DBHB, both utilising acetoacetyl-CoA.
• One manipulating the acetoacetate pathway and the other using 3-hydroxybutyrate-CoA (3HB-CoA) as an alternative intermediate (DBHB_B). The genes required for these two pathways were assembled into synthetic gene operons in silico (Figure 1)

Figure 1 - The synthetic DBHB producing operons designed. The four gene DBHB_A operon utilises genes taken from Clostridium acetobutylicum (thl, ctfA and ctfB) and Streptococcus dysgalactiae (bdhA). The thl gene in the DBHB_B pathway is the same as in the DBHB_A pathway but is followed by the phbB gene take from Cupriavdus necator and tesB from Escherichia coli.

Choosing our engineered DBHB production operon

• To determine if the production of therapeutic levels DBHB is achievable via either pathway in C. sporogenes and to inform which pathway to implement in our final production strain, shuttle vectors carrying each operon were designed.
• Both the acetate and hydroxybutyrate-CoA pathways would be assembled into a pMTL82151 shuttle vector by HiFi assembly, with four variant promoter regions upstream of the ketone production operon (KPO) to assess the different production profiles of DBHB that could be desirable (5).

Genomic integration of the synthetic pathway

• Without lab access, the most favourable KPO to integrate was ultimately decided by our modelling team, namely DBHB_A.
• Integration into the chromosome will allow stable expression of the pathway in our therapeutic strain without the need to maintain a plasmid vector by antibiotic.
• We designed RiboCas vectors that would facilitate the integration into the chromosome at the pyrE locus in C. sporogenes (6). The operon was designed with strong bidirectional terminators upstream (T_fad) and downstream (T_fdx) to isolate from chromosomal read through (Figure 2).

Figure 2 - Integration of the DBHB_A operon into the C. sporogenes chromosome at the pyrE locus, chosen as it is a known location for genomic insertions in Clostridia. Vectors to insert the pathway controlled by three promotor variants were designed: P_fdx (constitutive expression), P_ntnh (growth dependent expression), P_lac (lactose inducible expression).

Containment and biosafety strategy

As our project involves the delivery of genetically engineered microorganisms to the human gut, we needed to design a biosafety mechanism to prevent our engineered C. sporogenes strain escaping from the gut into the environment. Inclusion of a biosafety mechanism in our biotherapeutic strain of bacteria would help address concerns surrounding the biocontainment of synthetic organisms.

Our team explored three kill switch mechanisms during the selection process; lactose, synthetic amino acid and tetracycline.

• The lac repressor was a good candidate but would be useless as lactose is present in the gut.
• The synthetic amino acid method was very effective but also overly complex as we would have to modify all existing UAG stop codons so that it only targeted our chosen genes.
• The tetracycline system was a perfect candidate as the activating molecule is not present in the gut naturally and has been used successfully in Clostridia before.

We would implement this by genetically engineering C. sporogenes with the Tet system upstream of our target genes. We decided to target the genes spoIID, spoIVA and spoIIIAA as they are vital for sporulation. The Tet system is described below:

Figure 1. Diagram to show how the Tet repressor system we plan to implement as a biosafety control for our biotherapeutic strain works. In the top panel, anhydrotetracycline (ATC) is absent (as in the gut) and the Tet repressor (TetR) binds to the tet promotor upstream of the target gene and no expression of the target gene occurs. However, when ATC is present in the production process (as shown in the in bottom panel) it binds to TetR preventing it binding to the tet promotor and expression of the target gene can occur – in this case spoIVA.

Human Practices

We looked to explore the ethical and social implications our biotherapeutic product ‘NeuroTone’ could have for different publics, stakeholders and the wider society.

Integrating human practices from the start

To begin with, we had a workshop facilitated by our Human Practices supervisor to discuss the initial scope – and potential impacts, risks and benefits – of our project, identifying possible stakeholders and the publics who might be directly or indirectly affected.

We had several informal conversations with:

• industry representatives
• a medical professional
• an academic expert
• and a care provider

During these discussions, we explored opinions about the application of our project in the real world, technical issues we wanted to address, and social, ethical, and environmental aspects. From these interactions, we were able to learn more about the context in which our product would be developed and used, as well as the values and needs of end users and involved stakeholders.

Structural Modelling

The structural modelling subgroup was presented with the task to identify which pathway to produce DBHB would be most beneficial for our project and to suggest ways to improve DBHB production.

Model construction and improvement

Before beginning our analysis, we improved our structural model by:

• Standardising reactions by introducing reversible reactions.
• Adding molecular formulas to all known compounds.
• Adjusting equations, where relevant, to achieve a mass balance.
• Standardising the biomass production equation.
• Addition of pathways for the proposed mutants.

Identification of the best candidate

We used COBRApy and CellNetAnalyzer to perform flux balance analysis (FBA) and elementary mode analysis (EMA), respectively, to analyse the behaviour of:

• Wildtype,
• Mutant DBHB_A (acetoacetate pathway),
• Mutant DBHB_B (hydroxybutyrate-CoA pathway).

Figure 1: Results of our elementary mode analysis for the two mutants, DBHB_A (left) and DBHB_B (right), for modes supporting biomass and DBHB formation.

Based on its greater maximum ATP and DBHB productivity, growth yield and a higher number of supporting modes offering higher flexibility (see Figure 1), we decided that the mutant containing the DBHB_A pathway was the best choice to achieve our objectives.

Improvement of DBHB formation

To improve DBHB formation in mutant DBHB_A, we investigated three different scenarios:

• Assimilation of acids excreted by other bacteria in the gut
• Restriction of the mutant’s capability to excrete acids, Figure 2
• Knockout of ethanol formation

Figure 2: Flux balance analysis of mutant DBHB_A with constraint capability of acid excretion. The less acids that can be excreted, the more that flux is diverted towards the formation of DBHB.

Our results indicate that assimilation of acids and restriction of the mutant’s capability to excrete acids could increase DBHB production. Interestingly, an ethanol knockout would have less impact - our analysis suggests that DBHB formation is favoured over ethanol (and butanol) formation.

Dynamic Modelling

The dynamical model sub-group was tasked to investigate how the culture of our engineered C. sporogenes strain behaves in the gut and how administration of the drug can be optimised with respect to efficacy, tolerability and administration.

A kinetic model for the formation of DBHB in the gut

We build upon a previously developed model (7) which was developed to describe ABE fermentation in batch cultures of C. saccharobutylicum. The model parameters were adapted according to the results from the Nottingham iGEM team 2019. The ABE pathway as found in C. sporogenes including the synthetic acetoacetate pathway DBHB_A are shown in Figure 1.

Figure 1: The ABE pathway of C. sporogenes (black) with the addition of the DBHB_A pathway. Acetoacetate is formed using CoA-transferase CtfA/B from C. acetobutylicum (green) which is then converted by the action of 3-hydroxybutanoate dehydrogenase (3-BDH) (red) of S. dysgalactiae. R_i is the reaction identifier used in our kinetic model.

In order to model the growth of our DBHB_A and the production formation in the gut, we had to convert the existing batch-culture model into one that is better suited for an open system. The required additions included:

• regular uptake of glucose (growth controlling nutrient) to reflect administration of NeuroTone
• outflow terms for all compounds
• an additional (inducible) thiolase from C. acetobutylicum to increase flux towards acetoacetyl-CoA

Optimising the administration of NeuroTone

From our discussions with stakeholders (see Integrated Human Practices for more details), we learned that several aspects of the administration of NeuroTone should be optimised along with identifying measures to control the level of DBHB. Thus, we have conducted an investigation of key parameters of our model including:

• minimisation of frequency to make it easier for nurses and carers to administer NeuroTone,
• initial amount of spores,
• delayed induction of the DBHB operon,
• dosage of NeuroTone administered,

- importantly, without losing sufficiently high and stable DBHB levels.

Figure 2: Results from the optimisation of the administration strategy for NeuroTone. From left to right, the effect of different doses of NeuroTone, different initial numbers of spores, and induction of the DBHB operon at a later time.

Our analysis indicates that administration of NeuroTone every 48 hours is the best compromise between administration frequency and variation in DBHB levels.

Furthermore, the system approaches a stable limit cycle, which ensures its robustness against inevitable fluctuations in the frequency patients take NeuroTone. The adaptation of the gut microbiota to the engineered NeuroTone strain could benefit from a lower initial amount of spores.

Finally, the dosage of NeuroTone per capsule controls the average DBHB levels in the gut and, thus, allows regulation according to the individual patient's needs.

Outreach

Podcast

Over the summer, we recorded a series of podcasts – featuring other iGEM teams!

• Paris-Bettencourt
• King’s College London
• University of Warwick
• University of Southern Denmark

Making the most of our time at home, we were able to have some great chats with fellow iGEMers – talking about anything from debating breakfasts to dreaming about the fate of the Giant Jamboree!

Minecraft

Two of our members dedicated many hours of work to creating a Minecraft map of our own university campus. Building the visual landmarks of Trent and Portland Building, we created a storyline revolving around synthetic biology to educate and engage younger members of the community – perfect for a safe, stay-at-home learning activity(!)

Therapeutics Meet Up

On the 19^th September, we co-hosted a virtual conference open to all teams on the Therapeutics iGEM track. With the incredible help of the King’s College London iGEM team, we were able to create a space for therapeutics teams to collaborate and engage in the friendly, communal and open environment of the iGEM competition.

Tough Mudder for Charity

Recognising the importance of the issue we were dealing with, we decided that we should raise money for a charity putting their efforts into treating neurodegenerative diseases – we chose the Cure Parkinson’s Trust (CPT).

Taking on the extreme Virtual Tough Mudder Challenge, five of our team members embarked on a 12-hour challenge through the middle of the night. No surprises – it was tough! We had to run 5 miles every hour for the whole 12 hours, with trademark Tough Mudder challenges in between.

In the end, we were so proud to have raised a hugely generous £515 for CPT!

Team bonding to say the least.

Contributions

Despite the unprecedented circumstances, we realised the need for utilising and improving previous iGEM years' contributions along with adding to this ourselves.

As such, as part of our modelling research, we built upon the 2019 University of Nottingham's iGEM team model, improving and refining it. In terms of our contributions, we not only uploaded our structural modelling files on our wiki but we also made our MATLAB kinetic model code open access for future iGEM teams to utilise.

Pandemic or not, podcasts are a popular way of remotely gaining knowledge and passing time. For this reason, we decided to join the global trend and create an iGEM podcast discussing an array of topics that to interest people - such as dealing with a lockdown, working virtually as a team, conducting research remotely and of course with fun question and answer sessions.

We believe that such modes of communication and outreach are only getting more recognised over the years and hence it will be a powerful tool for future iGEM teams to interact with others. Allowing them not just to engage the public but to also establish valuable networks and partnerships with the teams and individuals across the world.

In order to make the process easier with limited resources and basic electronic items such as their phones, we made an invaluable podcast guide for future iGEM teams to follow with steps ranging from how to record all the way to the final editing. This should make producing a professional and impactful podcast attainable for all teams in a variety of circumstances.

Proposed Implementation

What is the Impact of NeuroTone?

• Innovative, non-invasive method of treating disease throughout the body.
• Powerful tool for delaying the onset of neurodegenerative disease.
• Maintaining quality of life, not just extending years.

Our Vision

The final vision for NeuroTone is a capsule containing spores of our novel biotherapeutic strain of Clostridium sporogenes, which may be prescribed to delay or prevent the onset of neurodegeneration in patients identified as at risk.

Individuals in this at risk group may be prescribed NeuroTone in a prophylactic manner at the critical time before irreversible damage to neurons has occurred and the symptoms of these diseases can manifest. Ideally, we would look to start treatment as soon as possible to have the most impact.

Safety

Our research has shown that increasing DBHB concentration in the blood could lead to complications for patients suffering with diabetes and kidney problems. We also need to make sure that NeuroTone does not react adversely with any other drugs, especially antibiotics. If we were to produce NeuroTone in the real world, we would need to go through years of robust drug trials in order to prove it is safe for use in patients.

How Others Will Use our Project

NeuroTone is not just an innovative way of delaying the onset of neurodegenerative diseases but is a novel way of approaching drug delivery. We envision others using the principles we have developed for NeuroTone to treat many other diseases via the gut. The microbiome is a fantastic target for drug delivery as it has access to almost every part of the body through the circulatory system and live therapeutic cultures of Clostridia provide a more reliable coverage of the selected drug, ensuring easy administration for patients and healthcare professionals.

References

1. GBD 2016 Neurology Collaborators (2019). Global, regional and national burden of neurological disorders, 1990-2016: a systematic study for the Global Burden of Disease Study 2016. The lancet, neurology, 18(5), 459-480.
2. Kashiwaya, Y., Takeshima, T., Mori, N., Nakashima, K., Clarke, K., Veech, R.L. (2000). d-β-hydroxybutyrate protects neurons in models of Alzheimer's and Parkinson's disease. Proceedings of the national academy of sciences of the united states of america, 97(10), 5440-5444.
3. Ota, M., Matsuo, J., Ishida, I., Takano, H., Yokoi, Y., Hori, H., Yoshida, S., Ashida, K., Nakamura, K., Takahashi, T., Kunugi, H. (2019). Effects of a medium-chain triglyceride-based ketogenic formula on cognitive function in patients with mild-to-moderate Alzheimer’s disease. Neuroscience letters, 690, 232-236.
4. Zhang, H., Yang, C., Zhou, W., Luan, Q., Li, W., Deng, Q., Dong, X., Tang, H., Huang, F. (2018). A pH-responsive gel macrosphere based on sodium alginate and cellulose nanofiber for potential intestinal delivery of probiotics. ACS sustainable chemistry and engineering, 6(11), 13924-13931.
5. Heap, J.T., Pennington, O.J., Cartman, S.T., Minton, N.P. (2009). A modular system for Clostridium shuttle plasmids. Journal of microbiological methods, 78(1), 79-85.
6. Cañadas, I.C., Groothius, D., Zygouropoulou, M., Rodrigues, R., Minton, N.P. (2019). RiboCas: a universal CRISPR-based editing tool for Clostridium. ACS synthetic biology, 8(6), 1379-1390.
7. Shinto, H., Tashiro, Y., Yamashita, M., Kobayashi, G., Sekiguchi, T., Hanai, T., Kuriya, Y., Okamoto, M., Sonomoto, K. (2007). Kinetic modeling and sensitivity analysis of acetone-butanol-ethanol production. Journal of biotechnology, 131(1), 45-56.

Acknowledgements

Our Advisory Team

Principal Investigators- Prof. Nigel Minton, Dr Andrew Dempster

Supervisors- Dr Raquel Rodrigues, Alexander Rawson, Dr Patrick Ingle, Dr Thomas Millat, Jacqueline Minton, Louise Dynes, Dr Eleanor Hadley Kershaw

Outreach Partners- The Cure Parkinson’s Trust, Tough Mudder, Alzheimer’s Society, GenScript

Our Sponsors- The University of Nottingham, Synthetic Biology Research Centre - Nottingham, The Wellcome Trust, Biotechnology and Biological Sciences Research Council, Engineering and Physical Sciences Research Council

iGEM Teams- KCL UK iGEM 2020 team, St Andrew’s iGEM 2020 team, Paris-Bettencourt iGEM 2020 team, Warwick iGEM 2020 team, Amsterdam iGEM 2020 team, Nottingham iGEM 2019 team.

Modelling Support- Dr Nicole Pearcy

Human Practices Support- Dr Edward Green/ Mr Ben Bradley, Dr John Morrant, Professor Kieran Clarke, Nicola Cook, Alice Hodson.

Wiki Support- Adam Bainbridge, Abigail Conner, James Abbott

Communications and Logistics- Cameron McCulloch-Keeble, Jess Wright, Nemira Zilinskaite.