Forget-Me-Not: a study of Alzheimer’s by the North Carolina iGEM team

As part of our partnership with iGEM, the biggest synthetic biology competition in the world, we are showcasing some of the exciting projects from teams that share our vision of synbio impacting positively on human health. This week, we've let the team from North Carolina State University (NCSU) take over our blog to share what they are working on. In this blog, they introduce their project: Forget-Me-Not and also cover the main ways they are approaching the design and build of their iGEM solution. 

Setting up our team - continuous feedback for success

One of the first key takeaways that we established from iGEM is the continuous feedback loop each team needs to put in place between the engineering cycle and the human practices (HP) cycle. The engineering cycle of design, build, test, and learn does not happen in isolation. The HP cycle involves identifying stakeholders, consulting communities, evaluating ethical and social impacts, and iterating based on feedback.

Secondly, iGEM informed our perspectives on biosecurity. Safety was an important design consideration in both of these cycles. We spent time creating an interdisciplinary team and examining our assumptions about emerging technologies such as CRISPR gene editing and Quantum Computing. We discussed the potential of off-target mutations, ran a public opinion survey, and researched worldwide regulations on gene therapy.

Moving forward in our scientific careers, the interplay between these cycles will remain at the forefront of our minds. We aim to approach every project with curiosity and compassion, seeking diverse perspectives to guide discovery in service of society. The lessons from iGEM about ethical STEM practice continue to shape us as researchers committed to meaningful innovation that transforms lives.

Our mission: Preemptive prevention of Alzheimer's disease

Alzheimer's disease (AD) inflicts a devastating toll, robbing millions of their memories, personality, and independence. As global lifespans increase, over 50 million people are projected to have dementia by 2050. 2023 has been a landmark year in AD therapeutics. Controversial new drugs like Lecanemab and Aducanumab offer a glimmer of hope by briefly slowing cognitive decline. However, with hefty price tags and limited efficacy, they remain out of reach for most patients.

Studies like the Finnish Geriatric Intervention Study demonstrate the power of early, multi-modal interventions in AD. Inspired by the growing burden of dementia and gaps in care exacerbated by COVID-19, we aimed to develop an affordable, preemptive solution.

By exploring links between the gut microbiome and the brain, we engineered a dual plasmid-induced CRISPR system that can be used to create engineered probiotics. Delivered at early stages of cognitive impairment, our synthetic biotic leverages the gut-brain axis to reduce neuroinflammation and amyloid plaque deposition. Quantum machine learning tools enabled diagnosis of early decline, allowing for prompt intervention.

Beyond combating AD, our project - which we named Forget-Me-Not - champions a paradigm shift in the American pharmaceutical complex, from reactive treatment to proactive prevention. The project demonstrates that preserving brain health is possible when we act early.

Learning outside the lab

Our human practices journey began by surveying AD stakeholders – caregivers, doctors, researchers, and patients. We heard repeatedly that research into neurodegenerative diseases had slowed during the pandemic. Caregivers also faced major challenges in accessing resources.
These insights guided our mission - developing both ethical guidance on emerging biotech and practical tools to empower caregivers.

On the ethics side, we authored an extensive neuroethics report analysing genetic engineering for treating neurological conditions. We considered complex questions around editing germline versus somatic cells and weighed concerns about ‘playing God’ against the potential to alleviate suffering from diseases like AD. By extensively consulting medical ethics experts, we aimed to shape a thoughtful framework for using new tools responsibly.

As a caregiver resource, we created a centralised health app that provides critical support for dementia patients and caregivers by addressing common challenges. Dementia patients often struggle to remember medical details, manage complex medication regimens, and track appointments, especially as cognition fluctuates day-to-day. A consolidated app allows patients and caregivers to access vital information like treatment plans, test results, educational materials, and memory journals in one place. We have created and translated memory journals in three languages for global access.

Our goal was to reduce inequalities by improving coordination and streamlining data sharing for caregivers. Sharing this resource with local ‘End Alzheimer’s organisations, showed the app decreased stress and time spent on logistics for caregivers. By pursuing lab work, ethics, and real-world tools in parallel, we gained a fuller perspective on the disease.

This cross-disciplinary approach stretched our thinking and deepened our commitment to compassionate science that serves people.

Methods, tools and techniques

Precision editing of the gut microbiome represents an exciting new frontier in synthetic biology. By reprogramming bacteria resident in the gut, we can alter human health outcomes. To pursue targeted microbiome engineering, our team developed a dual plasmid CRISPR-Cas9 gene editing toolkit.

Our system’s first plasmid contains the Cas9 nuclease, codon-optimized for expression across Clostridial species. This allows the editing machinery to function efficiently in different strain chassis. This plasmid also houses a riboswitch control system adapted from prior iGEM work (Nottingham 2020). The riboswitch precisely regulates Cas9 activity. The second plasmid houses the guide RNA, optimized at the design stage to limit off-target effects.

As a proof-of-concept, we used this toolkit to modify Clostridium butyricum, to increase butyrate production by altering key metabolic genes. Upregulating butyrate synthesis at the mild cognitive impairment stage helps manage AD's symptoms by reducing neuroinflammation and plaque buildup in the brain. To boost butyrate synthesis via our synthetic biotic, we harnessed the native Clostridial ketone body synthesis mechanism and engineered the acetone-butanol-ethanol pathway.

Beyond Clostridium butyricum, our modular dual plasmid platform enables targeted editing in diverse gut microbes. By altering gene function, we can reprogram the microbiome's metabolic output. This foundational CRISPR-Cas9 toolkit establishes a precision gut editing capability with broad applications. From enhancing short-chain fatty acid synthesis to degrading toxins, it empowers precise microbiome reprogramming. Our work helps expand access to this transformative technology.

Our synthetic biotic has to be delivered at the Mild Cognitive impairment stage of AD. To properly identify this, we used Quantum Computing to classify imaging studies. Our hybrid model achieved over 80% accuracy in identifying Mild Cognitive Impairment from a benchmark dataset.

To establish a prognostic model beyond our synthetic biotic, we used Quantum computing to identify acetylcholinesterase inhibitors as potential drug targets. For drug candidate screening, our quantum machine learning algorithm was able to identify known acetylcholinesterase inhibitors with 78% accuracy from a large compound library. 

Big picture

Putting together the different components of our project, our team recognised the need for a paradigm shift in the American pharmaceutical complex, from reactionary treatment to proactive prevention. A systematic review of precision medicine approaches for AD analyzed key pillars like prevention, diagnosis, prognosis and treatment. This framework guided the multifaceted nature of our project.

Additionally, feedback from over 70 AD stakeholder interviews helped shape critical improvements:

• For our machine learning models, we instituted federated learning - keeping data decentralised and local to prevent misuse.
• In our probiotic system, we engineered auxotrophy requirements, so the gene editing machinery only functions in the presence of unnatural amino acids. This prevents horizontal gene transfer between gut bacteria.
• We partnered with community organisations, like Leeza’s place, to create culturally relevant educational content and memory journals translated into different languages. This makes resources more accessible to underserved groups.

By integrating human-centered findings with scientific capabilities, our project evolved to address ethical and social priorities. Our team aims to develop solutions that serve the whole person, not just the disease.

...And beyond!

At its core, Forget-Me-Not embodies the values and mission of the iGEM competition. Since its inception, iGEM has been committed to responsible advancement of synthetic biology to benefit society. Our team strongly believes in this purpose.

We develop tools to enable ethical research and create resources to empower underserved communities in the AD realm and gene therapy research. We aim to exemplify iGEM values of compassion, curiosity, and collaboration

Our project contributes to the global effort to shape biology in service of humanity. As members of the worldwide iGEM community, we will carry this spirit of discovery and service forward in all our future endeavours.

Pioneering as a team

This project would not be possible without the continued support of our PI, Dr. Eric Rotenberg. Neha Suresh (wet lab) pitched the idea of beginning an iGEM team at various courses across NCSU. After gauging interest among NC State peers, she began assembling a diverse team. We range from biologists to programmers, tackling complex concepts from varying perspectives. Leveraging the Quantum computing resources on campus, we brought Ramachandran Srikanthan on board to lead the dry lab team in modeling Alzheimer’s disease. Seeva Cherukuri led human practices and stakeholder engagement throughout the project cycle. All three team leads created organisational processes to improve communication and transparency. Early team meetings involved spirited debates on project direction, balancing ambitious ideas with feasibility. By scheduling calls with previous iGEM teams and attending Synbiobeta 2023, we gained corporate sponsors and mentors who were invaluable at every step of our journey. 

References:

1. Qian XH, Xie RY, Liu XL, Chen SD, Tang HD. Mechanisms of Short-Chain Fatty Acids Derived from Gut Microbiota in Alzheimer's Disease. Aging Dis. 2022 Jul 11;13(4):1252-1266. doi: 10.14336/AD.2021.1215. PMID: 35855330; PMCID: PMC9286902.
2. Liu J, Sun J, Wang F, Yu X, Ling Z, Li H, Zhang H, Jin J, Chen W, Pang M, Yu J, He Y, Xu J. Neuroprotective Effects of Clostridium butyricum against Vascular Dementia in Mice via Metabolic Butyrate. Biomed Res Int. 2015;2015:412946. doi: 10.1155/2015/412946. Epub 2015 Oct 7. PMID: 26523278; PMCID: PMC4615854.