Project Description
Defining the Problem
Worldwide, upwards of 55 million people are currently living with dementia with an average of 10 million people being diagnosed each year (World Health Organization, 2021). Of these, approximately 60-70% are Alzheimer's disease (AD) cases (World Health Organization, 2022). AD is a progressive neurological condition (DeTure and Dickson, 2019) and is the most commonly diagnosed type of dementia (Silva et al., 2019). By 2050, it is estimated that the global population of people living with dementia will be approximately 139 million (Alzheimer's Disease International, 2022).
Robinson et al. (2015) and The Alzheimer's Association (2020) describe dementia as a clinical syndrome that encompasses difficulties with:
- Memory loss and thinking
- Problem-solving or language
- Increased confusion and disorientation
- Changes in mood
- Difficulty to carry out tasks
- Depression
Dementia develops when the brain is damaged by various underlying diseases, including AD (van der Flier and Scheltens, 2005), and symptoms develop gradually over many years, increasing in severity, resulting in AD being a time-limiting illness (Ashworth, 2019). More information regarding AD can be found on our pathophysiology of AD page. A study by Pascoal et al. (2021) suggests that it is an interaction between neuroinflammation and amyloid pathology that is required to unleash tau propagation that eventually leads to widespread brain damage and cognitive impairment. In other words, tau tangles, plaques and neuroinflammation are required altogether to lead to progression. Tau tangles and plaques itself are not enough to lead to this progression.
According to the European Federation of Pharmaceutical Industries and Associations (2020), 25% of 65 year olds and above in Europe are predicted to be diagnosed with a type of dementia by 2030 (Figure 2). In the UK, the number of people estimated to have a form of dementia will reach 1 million in 2024 and 1.2 million people by 2030 (Wittenberg, 2019).
AD does not only affect the patient but also has implications for family members and carers. Approximately 10 million families globally undergo extreme stress from witnessing their loved ones experience their progressive condition daily (Cuyler, 2019). Additionally, there are also several major challenges surrounding the quality of palliative care due to inadequate funding within the health and social care sector (O'Dowd, 2015), which is not suitable for the current upward projection of the ageing population. Consequently, this emphasises the need for a progressive approach to the psychological and socioeconomic impact AD has.
Project Overview
At Symemco Therapeutics, we aim to develop a disease-modifying therapeutic to stagnate the neuroinflammatory progression of mild to moderate stage AD. We plan to execute this by genetically engineering Escherichia coli to produce high yields of the polyphenol, pterostilbene.
We believe pterostilbene has the potential to be an effective treatment for AD as it has anti-inflammatory properties which have shown to down-regulate neuroinflammation associated with AD progression in mice models (Wang et al., 2020) and rodent cell lines (Li et al., 2018). In doing so, we hope to help those living with AD maintain the relationships and connections they have with themselves, their loved ones, and the world.
Our novel synthetic biology solution
Utilising synthetic biology to tackle this urgent local and global issue is at the forefront of Symemco Therapeutics’ vision and mission. As opposed to other methodologies, synthetic biology provides many advantages, including the efficiency of drug target discovery, ensuring high bioavailability, and efficacy of our therapeutic (Xie et al., 2020). Synthetic biology also has the potential to impact drug manufacturing on a global scale, providing a high performance and sustainable method of production (World Economic Forum, 2021). By genetically engineering E. coli to produce a viable therapeutic concentration of pterostilbene to stagnate mild to moderate AD neuroinflammatory progression, we hope to create an impact globally through synthetic biology on AD therapeutics, as well as other neurodegenerative diseases, such as Parkinson’s disease.
Comparative to currently available treatments, we will be targeting a different key pathophysiological hallmark of mild stage AD, the NLRP3/caspase-1 neuroinflammatory pathway (Li et al., 2018), to delay the neuroinflammatory progression of mild to moderate AD (Figure 3). Literature has suggested that pterostilbene targets the NLRP3 inflammasome by binding to the ATP hydrolysis site, and thereby prohibits the NLRP3 inflammasome's mechanism of action within neuroinflammation related to AD (Chen et al., 2021). This is an intracellular protein complex consisting of: the nod-like receptor protein 3 (NLRP3) scaffold, the adaptor protein (also referred to as the apoptosis-associated speck-like (ASC) protein) containing a caspase recruitment domain and procaspase-1.4 (Moossavi et al., 2018).
Results from literature have demonstrated that pterostilbene also inhibits amyloid-β-induced neuroinflammation in microglia. Pterostilbene achieves this by significantly reducing pyroptosis and cytokine secretion in the NLRP3/caspase-1 inflammasome pathway (Li et al., 2018). This is dissimilar to existing treatments, such as donepezil and memantine, that target the palliative treatment of AD rather than its underlying pathophysiology. Nonetheless, there are currently 143 agents in clinical trials in the US alone as of January 2022, of which 83.2% were disease-modifying therapies (Cummings et. al., 2022), implying the direction of AD therapeutics is heading towards the targeting of its underlying pathophysiology.
Project Goals
- Establish that our novel biosynthesis pathway of producing pterostilbene reaches and surpasses the maximum yield stated in literature to be able to produce economical therapeutic concentrations of pterostilbene through in silico modelling and genetically engineered E. coli.
- Develop a comprehensive business plan, assessing the feasibility and the potential implementations of our project, in order to open the opportunity to expand and advance Symemco Therapeutics after the iGEM competition has concluded.
- Centre accessibility within all aspects Symemco Therapeutics by creating guides for a range of components throughout the subgroups of our project. In addition, we aim to promote synthetic biology and raise awareness of Alzheimer’s disease through our social media platforms and educational outreach workshops, whilst using a dyslexic and colourblind friendly theme.
We have also determined separate subgroup goals that we intend to meet through specific aspects of our project. These are detailed below.
Within the dry lab team, we aim to model gene expression in E. coli through a deterministic model that would provide us with indication as to what would be the optimal plasmid copy number and promoter strength to achieve the highest possible pterostilbene yield considering E. coli's metabolic needs for L-tyrosine.
Achieving this will involve three stages. Firstly, we will be modelling E. coli metabolic needs for L-tyrosine to find out how much L-tyrosine can be used for pterostilbene synthesis per unit of time. We will then model enzyme kinetics of the pterostilbene production pathway. Secondly, given the amount of L-tyrosine available per unit of time, we will determine the enzyme concentration that catalyses pterostilbene synthesis reactions produced by E. coli without overburdening the bacteria. Lastly, we will be modelling the gene expression and determining the optimal promoter strength and plasmid copy number given the information obtained in the first two stages.
The model, in our case, will be based on the law of mass action and will provide information based on which optimal decisions regarding genetic modifications in the E. coli will be made. We also aim to determine oral bioavailability of pterostilbene and blood-brain barrier permeability in order to prove the feasibility of our manufacturing approach in relation to the dosage per patient required. This will be determined through implementation of already existing models and guided by literature sources on pterostilbene properties.
By utilising synthetic biology in our manufacturing method, we intend to produce the therapeutic dose of pterostilbene. Recent genetically engineered approaches using E. coli, have resulted in a maximum yield of 80.4mg/l of pterostilbene (Yan et al., 2021). Through our novel biosynthesis method using mutated enzymes, we plan to surpass this maximum yield to produce therapeutic concentrations of pterostilbene. These mutated enzyme would be tyrosine ammonia lyase enzyme from Rhodotorula glutinis, 4-coumarate coenzyme-A ligase from Arabidopsis thaliana, and stilbene synthase and resveratrol-O methyl transferase from Vitis vinifera. All four enzymes have been modified from their wild type sequence according to literature, which identifies mutations increasing their catalytic capacity (Yan et al., 2021).
By developing an understanding of the pathophysiology of AD we aim to gain clarity on the mechanisms of neuroinflammation associated with AD. Subsequently, we aim to use this information to establish the optimal therapeutic dose of pterostilbene and determine specifically where, when and how our therapeutic is proposed to target AD. Moreover, we intend to expand our knowledge of the stages of AD and the hallmarks of the disease to ensure the research we are conducting in the laboratory and our intended application of pterostilbene is up to date with the current knowledge surrounding AD generally. To aid our understanding, we plan to reach out to scientists and clinicians where possible to further develop our understanding of AD as a whole.
Through delivering numerous interactive workshops and seminars to students aged 14-18, we aim to foster an open dialogue between synthetic biology and the Middle Years Programme (MYP) and International Baccalaureate (IB) syllabi. In relation to our overall project, we will highly focus on the pathophysiology and current treatment of neurodegenerative disease, as well as our ability to genetically engineer E. coli bacteria. This greatly links to recent additions to the IB Biology curriculum regarding biotechnology and the engineering of E. coli to produce insulin for diabetic patients. In addition to this, we also wish to introduce students to the multidisciplinary aspects of iGEM and the synthetic biology and biotechnology industry as a whole. Within this, we plan to provide students with model business plans and the opportunity to present a drafted pitch deck. Further in the future, we will distribute the building blocks to host a week-long biohackathon to several global institutions of which we have already received large interest. The competition will follow a smaller-scale iGEM format and hopefully increase student and institution application for upcoming years.
Project Inspiration
In the development of our project, we have taken inspiration from a range of previous iGEM teams, namely the 2014 Massachusetts Institute of Technology (MIT) team, the 2018 McMaster university team, the 2019 Jiangsu High School team, and the 2013 Uppsala team.
In 2014, the MIT iGEM team designed a conjoined diagnosis and treatment project based on the aggregation of amyloid-β oligomers, a hallmark of AD. Their project highlighted current issues with both the diagnostics and treatment of AD. Patients often show characteristics of AD but also mask their symptoms. Additionally, there are also overlaps in symptoms between neurological diseases such as Parkinson’s disease. The 2014 MIT team identified that current treatments were often ineffective at treating late stage AD as the disease had spread too far, however, there was promise that early stage detection and treatment could be promising. Another previous iGEM team, the McMaster university team in 2018 also focused on AD and like MIT, they looked at the amyloid-β plaque formations that characterise AD. In one of their outreach interviews, McMaster iGEM spoke to a researcher that expressed concerns with the amyloid-β hypothesis. Additionally, the 2019 Jiangsu High School team also looked into developing an anti-AD drug, which focused on the autophagic flux in SH-SY5Y cells. However, no previous teams have focused specifically on the NLRP3/caspase-1 neuroinflammatory pathway, and alongside the promising research on caspases in the treatment of AD (Dhani et al., 2021), we saw an opportunity to utilise synthetic biology to work on a therapeutic that targets this area within AD.
The team also took inspiration from the 2013 Uppsala team’s use of ODE’s to model the yield of specific metabolites. We have improved on this model by making use of specified software to produce more accurate estimates for yield as well as account for variables such as metabolic strain of the introduced plasmids on the E. coli.
We have also taken inspiration from iGEM teams tackling the problem of inefficient resveratrol production that have introduced the biosynthesis of resveratrol into different microorganisms. The 2008 Rice University team introduced a 4CL::STS fusion protein into yeast, carried on in 2013 by the Bordeaux and Uppsala teams in E. coli. Consequently, these teams have similarly approached the detection of resveratrol production by high-performance liquid chromatography, with some further verifying its identity by mass spectroscopy. The success demonstrated in their experiments, which share most of the pathway enzymes, has inspired us to take this well-characterised resveratrol synthesis pathway further for pterostilbene biosynthesis in E. coli. The 2013 Uppsala team also designed and successfully sequenced and cloned a biobrick composed of two enzymes, 4-coumarate ligase (4CL) and Stilbene Synthase (STS) to produce resveratrol, an important precursor in the metabolic pathway of pterostilbene synthesis. Taking inspiration from this team and from further research in literature we identified two possible mutations to the wild type AT4CL enzyme used by Uppsala that would further optimise the activity of the enzyme by increasing active site substrate binding and thus catalysis.
References
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