DESCRIPTION

Project Brief

Epilepsy Shield: BHBio ＆ EEGSense

According to the 2024 statistics from the World Health Organization (WHO), there are approximately 50 million epilepsy patients worldwide. The ketogenic diet therapy is relatively effective in treating epilepsy by promoting the body’s ketone body synthesis; However, long-term adherence to the ketogenic diet may lead to side effects such as obesity and ketonuria.

To avoid the side effects of the ketogenic diet, we adopt synthetic biology approaches to synthesize ketone bodies for epilepsy treatment. Specifically, we designed a pathway for producing the ketone body β-hydroxybutyrate (BHB) in Escherichia coli Nissle 1917 and incorporated a dynamic regulation module into the system. The engineered bacteria are encapsulated in hydrogel to form capsules. After oral administration by patients, these capsules colonize the intestinal tract and synthesize BHB to treat epilepsy. Additionally, we developed an intelligent epilepsy monitoring system: a concealed behind-the-ear device (cEEGrid) collects the patient’s electroencephalogram (EEG) signals, which are then transmitted via Bluetooth to a receiving and delivery module. After processing by the software, the risk of epileptic seizures is visualized for clinical reference.

This project aims to provide new insights into the prevention and treatment of epilepsy through synthetic biology methods, with a focus on caring for epilepsy patients.

Fig1. Epilepsy Shield: BHBio ＆ EEGSense

Epilepsy Shield

The term "Shield" signifies defense, protection, and resistance—and our project aims to establish a biomedical barrier both inside and outside the human body. Internally, we have engineered bacteria to help the body build a "Shield": these bacteria produce β-hydroxybutyrate (BHB) to resist epilepsy. Externally, we have designed a concealed behind-the-ear device to serve as a second "Shield": it continuously monitors signals over a period of time and provides an assessment of seizure risk based on the frequency of abnormal EEG (electroencephalogram) patterns detected. This dual internal-external shield effectively resists, reduces, and even prevents the "attacks" of epileptic seizures, alleviating the harm and distress caused to patients by such seizures.

Before Finalizing the Research Topic

Prior to confirming our research topic, we conducted extensive interviews and surveys both online and offline across different regions of China. We visited campuses, communities, and public spaces, conducting 9interviews over a week with 82 individuals from diverse backgrounds, age groups, and occupations. During the interviews, we asked about the issues people are most concerned about in today’s society and the troubles they face in daily life. Respondents mentioned topics such as health, dietary health, and environmental protection—among them, 51 interviewees (approximately 74.39%) stated that their top concern was health and well-being. This led us to launch a discussion focused on health-related issues.

Brainstorming to Define the Topic

All team members collaborated in a brainstorming session to determine our research focus. During this process, several members shared experiences of relatives, neighbors, or acquaintances living with epilepsy. Others noted that historical figures and celebrities—such as Julius Caesar, painter Vincent van Gogh, and literary giant Fyodor Dostoevsky—also suffered from epilepsy and endured great hardship due to the condition. These stories and experiences deeply resonated with us.

In response, we conducted in-depth research, including interviews, literature reviews, and market analysis. From the findings, we discovered a key distinction between epilepsy and other diseases: epilepsy disproportionately affects low-income populations and children. Due to the difficulty of completely curing epilepsy, patients’ daily lives are severely disrupted, and their quality of life is significantly reduced. Additionally, society harbors widespread misunderstandings, prejudices, and discrimination toward epilepsy patients and their families. We witnessed the dual physical and psychological suffering endured by patients and their loved ones, and we also identified numerous flaws in existing epilepsy treatments that urgently need to be addressed.

Refining the Research Direction

After deciding to focus on epilepsy treatment, we outlined several potential directions for refinement:

1.Targeting neurotransmitters and their receptors

2.Improving the ketogenic diet

3.Applying CRISPR gene-editing technology

4.Studying the mechanisms of epileptic seizure termination

Subsequent literature reviews revealed critical limitations of these approaches:

For neurotransmitters:While interfering with neurotransmitter-receptor binding can treat epilepsy, long-term use harms the nervous system and may trigger other neurological disorders.

For CRISPR gene editing:The technology is costly, and its effects are irreversible. A single operational error could cause permanent harm to patients.

For seizure termination mechanisms: Once an epileptic seizure begins clinically, the window for intervention is extremely narrow (measured in seconds), and significant individual differences make timely and effective termination impossible.

Among the various epilepsy treatments, theketogenic dietstood out to us. Its mechanism involves maintaining a long-term high-fat, low-carbohydrate diet to inhibit hepatic gluconeogenesis. This forces the liver to activate fatty acid β-oxidation, producing ketone bodies that supply energy to the body. Ketone bodies act through multiple synergistic mechanisms to suppress abnormal neuronal excitability. Notably, the ketogenic diet can overcome drug resistance and is effective for various types of epilepsy. We were particularly drawn to this method—since it relies on the body’s own ketone body production to exert long-term effects on the nervous system and reduce neurological damage—and we sought to further improve it.

Market Analysis & Project Concept

Currently, medications dominate the epilepsy treatment market, accounting for approximately 70% of market share. While other treatment methods are gradually gaining traction, the overall market size for epilepsy treatment is growing slowly. However, there are very few—if any—products on the market that can replace the ketogenic diet for epilepsy treatment. The limited ketone body products available are primarily marketed for fitness purposes and are expensive, priced at 1,000 to 2,500 yuan per kilogram. If used as a substitute for the ketogenic diet, the required dosage of ketone bodies would be extremely high, leading to prohibitively costly treatment.

Based on the above observations, we conceived and designed engineered bacterial capsules encapsulated in hydrogel—a low-cost, low-risk approach that offers a new perspective for epilepsy treatment.

About Epilepsy

Pathogenesis

The core pathogenesis of epilepsy lies in the sudden, excessive, and synchronized abnormal electrical discharge of a group of neurons in the brain. This abnormality arises from the disruption of the balance between excitatory and inhibitory signaling systems in the brain, often caused by damaged ion channels and abnormal functions of neurotransmitters and their receptors (e.g., reduced levels of the inhibitory neurotransmitter GABA or excessive amounts of the excitatory neurotransmitter glutamate). Abnormal electrical activity originates from a local "epileptic focus" and then rapidly synchronizes and spreads through neural networks—like an "abnormal electrical storm" in the brain—ultimately triggering transient functional impairments that manifest as diverse forms of epileptic seizures. Epilepsy has numerous triggers, such as genetics, abnormal brain structure, parasitic infections, brain tumors, and traumatic brain injury. However, the cause of epilepsy remains unknown in a large number of patients worldwide.

History

Epilepsy is widely recognized as one of the oldest diseases in the world, with written records dating back to 4000 BCE. For dozens of centuries, people have continuously contributed to the fight against epilepsy.

Hippocrates, an ancient Greek physician, described the symptoms of epilepsy in his famous treatise On the Sacred Disease and argued that epilepsy was not a sign of demonic possession or divine punishment.

John Hughlings Jackson, the founder of modern epileptology research, was the first to propose that epilepsy is caused by "sudden, excessive, and rapid electrical discharge of neurons in the cerebral cortex." By carefully observing clinical symptoms to reverse-infer the location of brain lesions, he put forward the concept of "Jacksonian epilepsy."

In 1929, Hans Berger (1873-1941) invented the human electroencephalogram (EEG). This was a revolutionary breakthrough in the history of epilepsy research, enabling doctors to directly record and visualize abnormal electrical activity in the brain and providing an objective basis for the diagnosis, classification, and lesion localization of epilepsy.

From the second half of the 20th century to the present, with the continuous development of antiepileptic drugs (AEDs), the precise diagnosis of neuroimaging (e.g., MRI), and the rapid advancement of neuromodulation technologies such as vagus nerve stimulation (VNS), we believe that one day we will conquer epilepsy!

Current Status

People with epilepsy face a threefold higher risk of premature death compared to the general population. In low-income countries, three-quarters of epilepsy patients do not have access to the treatment they need.

Epilepsy is one of the most common neurological disorders globally, affecting approximately 50 million people worldwide. At any given time, 4 to 10 out of every 1,000 people require treatment for epilepsy. Compared to the general population, epilepsy increases the risk of premature death by three times. Nearly 80% of people with epilepsy live in low- and middle-income countries, and in many regions, epilepsy patients and their families face stigma and discrimination.

Given its widespread impact and severe effects on patients’ lives, our team is committed to developing safer and more effective treatment methods using knowledge of synthetic biology. We aspire to bring hope to patients suffering from epilepsy.

Fig 2. Global Statistics on Drug-Resistant vs. Drug-Responsive Epilepsy Fig 3. Proportion of Different Patient Types Fig 4. Comparison of Employment Rates among Epilepsy Patients in Different Country Types

Treatments

Currently, the main treatment methods for epilepsy include medication therapy, surgical therapy, neuromodulation therapy, and ketogenic diet therapy.

Table 1. Comparison of Epilepsy Treatment Methods

Treatment method	Advantages	Disadvantages
Drug Treatment	Effectie for about 70% of patients convenient to use, and easy for 1ndividualized adjustment	About 30% of epiepsy patients respond poorly to existng anti-eileptic drugs and there is long-term dependence
Surgical Resection	Surgicalresection can completely eliminate epilptic seizure,and about 60-70% of paitents achieve no seizures after surgery	Has surg ical risks, such as common compliations of craniotomy and high economic cost
Neurom odulation	Does not damage brain tissue,the equipments reversible and adjuetable	only reduces the frequency of seizures and does not completely cure;invasvie opreations have risks and the economic burden is high
Ketogenic Diet	can avoid drug side effects and surg ical risks,and can be effective for a long time	has side effects,will affect the card to vasular system in the long term,and cause grow thretardition in children

1、Medication Therapy

Medication therapy is the most common treatment for epilepsy. It works by administering drugs to inhibit the abnormal electrical discharge of neuronal cells, thereby reducing epileptic seizures. Currently, antiepileptic drugs (AEDs) are categorized into first-line, second-line, and third-line medications.

First-line drugs are the primary treatment choice and are often specific for epilepsy with certain symptoms. Widely used first-line drugs include Levetiracetam, Lamotrigine, Valproate, and Oxcarbazepine. The annual treatment cost is approximately $200 to $1,000 for generic versions, while brand-name drugs can cost as much as $5,000 to $12,000 per year.

Second-line treatment should be attempted as early as possible when first-line drugs are ineffective. It is important to note that there is no clear boundary between first-line and second-line treatments; sometimes, second-line treatment also involves combinations of first-line drugs. The annual cost for generic versions is roughly $500 to $3,000, and the cost increases significantly if brand-name drugs are used.

Third-line drugs (anesthetics) are typically used for patients with persistent or refractory epilepsy to control seizures and prevent damage to the brain and other organs. The cost of third-line drugs is highly correlated with the frequency of the patient’s seizures.

2、Surgical Therapy

Surgical therapy is suitable for pediatric epilepsy, epilepsy associated with tumors or vascular lesions, etc. It is generally divided into resective surgery and corpus callosotomy. The initial cost ranges from approximately $50,000 to $150,000 or even higher, with additional subsequent costs for nursing and rehabilitation.

Resective surgery is intended for patients with focal epilepsy. Its goal is to remove well-defined localized epileptic foci, eliminating the area of abnormal electrical discharge that triggers seizures and thereby terminating epileptic episodes.

Corpus callosotomy is used for patients who cannot benefit from lesion resection and those with generalized seizures. It involves cutting the nerve fiber bundle (corpus callosum) that connects the left and right cerebral hemispheres, blocking the bilateral spread of abnormal electrical discharge to inhibit seizures.

However, surgical therapy causes irreversible trauma and carries inherent risks of craniotomy, such as postoperative infection.

3、Neuromodulation Therapy

Neuromodulation therapy is effective for patients with epilepsy where the epileptogenic focus is difficult to locate and for those with various seizure types, with particularly good efficacy in pediatric patients. The cost of implanting the device ranges from approximately $30,000 to $50,000. This technique involves placing special electrodes deep in the brain to deliver electrical stimulation to specific regions (neural nuclei). A small stimulator is implanted under the skin of the chest; after the intracerebral stimulation electrodes are precisely positioned in specific deep-brain regions, they are connected to the stimulator via a thin wire, allowing electrical stimulation to be delivered according to a programmed protocol. Compared with surgery, this technique has the advantage of not requiring the resection or destruction of brain tissue. Various stimulation parameters can be adjusted until the optimal stimulation settings are found, and the device can be turned off promptly if adverse reactions occur. However, this treatment still causes trauma, the implanted devices are expensive, and long-term maintenance is required.

About Ketogenic Diet

Compared with other treatment methods, our team has focused on the ketogenic diet therapy, which demonstrates more obvious advantages.

Principle

The ketogenic diet (KD) is a special dietary pattern, mainly characterized by the intake of high fat (70%), moderate protein (25%), and low carbohydrates (5%). Under this dietary state, fat serves as the main source of calories, and the human body will enter a ketogenic state, producing ketone bodies such as β-hydroxybutyrate (BHB) that act as energy-supplying substances to provide energy for the body.

Fig 5. Dietary Structure of the Ketogenic Diet Fig 6. Mechanism of Action of Ketogenic Diet

The ketogenic diet therapy is more routine and easy to integrate into patients' daily lives. It is particularly suitable for patients with refractory epilepsy who have poor response to medication, as well as for children. In addition, the ketogenic diet has good therapeutic effects: clinical studies show that approximately half of pediatric patients experience a significant reduction in seizures after adhering to the ketogenic diet, and some even achieve a seizure-free state. However, it also has drawbacks such as side effects and poor compliance. Based on the above reasons, our goal is to explore ways to improve the ketogenic diet to help epilepsy patients.

Fig 7. Sustained Therapeutic Effect of Ketogenic Diet

Advantages

We focus on the ketogenic diet (KD) specifically for the following four reasons:

1、Compared with other treatment methods, the ketogenic diet can effectively overcome drug resistance and has a good therapeutic effect on drug-refractory epilepsy;

2、The ketogenic diet works through the combination of multiple pathways and mechanisms, making it effective for various types of epilepsy;

3、The ketogenic diet relies on the body’s own ketone bodies to act on the nervous system, resulting in a stable effect. Over the long term, it can reduce damage to the nervous system;

4、The ketogenic diet is more integrated into daily life, more acceptable to the general public, and can avoid drug side effects, drug dependence, and surgical risks. It can be said that the ketogenic diet is currently a preferred method for treating refractory epilepsy.

Research on the Ketogenic Diet for the Treatment of Other Diseases

1、Malignant tumors: Through its high-fat and low-carbohydrate dietary structure, the ketogenic diet can lower the level of circulating blood glucose. Under conditions of glucose deficiency, the proliferation ability of cancer cells is significantly reduced, thus controlling cancer cells. In addition, the ketogenic diet greatly increases the level of β-hydroxybutyrate (BHB), which can reverse the gene expression pattern of tumor cells to that of non-tumor cells, normalize cancer cells, and achieve a curative effect.

2、Obesity: Fatty acids in the ketogenic diet can suppress appetite and reduce patients’ desire to consume high-calorie foods. At the same time, under ketosis, the body uses fat instead of glucose for energy supply, consuming the body’s existing fat accumulation and thereby alleviating obesity.

3、Parkinson’s disease: The ketogenic diet can exert neuroprotective effects on Parkinson’s disease through a variety of mechanisms, including inhibiting neuroinflammation, resisting oxidative stress, regulating intestinal flora, improving mitochondrial function, enhancing brain metabolism, maintaining glucose concentration, and regulating autophagy.

About BHB

Molecular Characteristics

Ketone bodies (β-hydroxybutyrate, BHB) are derivatives of 4-carbon short-chain fatty acids and small lipid-derived molecules. They exist in two stereoisomeric forms: the D-form (biologically active) and the L-form (inactive). Chemically, they are present in three main forms: β-hydroxybutyric acid, β-hydroxybutyrate ester, and β-hydroxybutyrate salt, among which the anionic moiety is responsible for the therapeutic effect. Ketone bodies are not normal products of human metabolism. Instead, they are small molecules generated from fat breakdown to supply energy to tissues when glucose is scarce (e.g., during fasting or prolonged exercise). As small polar molecules, BHB is highly soluble in water and blood. With the assistance of several monocarboxylate transporters (including MCT1 and MCT2), it can cross the blood-brain barrier to reach the brain, thereby regulating brain activity.

Fig 8. Schematic Diagram of the Structure of Ketone Body β-Hydroxybutyrate (BHB)

Endogenous Production Pathway

In the mitochondria of hepatocytes, acetyl-CoA undergoes a reverse reaction under the action of thiolase to generate acetoacetyl-CoA. Acetoacetyl-CoA then produces acetoacetic acid under the catalysis of HMG-CoA synthase. Acetoacetic acid can be reduced under the catalysis of dehydrogenase to form the ketone body D-β-hydroxybutyrate (BHB). Mechanism of Action in Epilepsy Treatment Numerous studies have confirmed that BHB is the key molecule responsible for the main therapeutic effects of the ketogenic diet.

Mechanism 1:BHB can enhance gamma-aminobutyric acid (GABA) levels and the GABA/glutamate ratio in the brain, thereby inhibiting epileptic activity.

Mechanism 2:Neuron-specific enolase (NSE) levels in the blood are an important marker of brain damage. BHB can increase the expression level of NSE and decrease the expression level of glial fibrillary acidic protein (GFAP). This indicates a reduction in the number of damaged neurons, demonstrating that BHB exerts a protective effect on neurons.

Mechanism 3:Studies have shown that BHB treatment prolongs the latency of pilocarpine-induced epileptic seizures in mature mice, suggesting that BHB—one of the ketone bodies—may have a direct anticonvulsant effect.

Mechanism 4: Research has found that BHB can also activate hydroxycarboxylic acid receptor 2 (HCAR2). This may confer neuroprotective effects through the production of prostaglandin D2 (PGD2). Additionally, BHB promotes the recycling of synaptic vesicles, ensuring the continuous supply of the inhibitory neurotransmitter (GABA), while stabilizing the release of the excitatory neurotransmitter (glutamate). This reduces abnormal electrical discharges, ultimately achieving the effect of treating epilepsy.

Studies have revealed that after oral administration of ketone bodies, absorption occurs rapidly: blood ketone concentrations quickly reach a peak and then decline sharply, forming a "pulsatile" or "roller-coaster-like" pharmacokinetic curve. Such blood ketone levels are unstable. For disease treatment, a stable concentration in the body is required to achieve therapeutic effects. In contrast, endogenously produced ketone bodies can stabilize the amount of ketone bodies in the body at the required level, enabling long-term effective treatment.

The Limitations of the Ketogenic Diet

Side Effects

Although the ketogenic diet has unique advantages that cannot be replaced by other treatment methods, the traditional ketogenic diet therapy alters patients’ dietary structure. Maintaining a long-term high-fat, low-carbohydrate dietary pattern can lead to several adverse effects. For instance, during the initiation phase, reduced dietary fiber intake and increased digestive burden on the body may cause symptoms such as gastrointestinal discomfort, infectious diseases, and hypoproteinemia. During the maintenance phase, uneven nutrient distribution and insufficient energy/calorie intake can result in mild but persistent metabolic acidosis, with associated adverse effects including slowed growth, progressive loss of bone calcium, kidney stones, and iron deficiency. For pediatric patients, these effects may affect their normal growth and development to a certain extent. For many patients with other underlying conditions (e.g., hypertension, heart disease), the restriction on excessive fat intake means they have to forgo this otherwise advantageous treatment method.

Fig 9. Side Effects of the Ketogenic Diet Therapy

Low Adherence

The therapeutic effect of the ketogenic diet is highly dependent on patients’ strict compliance with the dietary plan. Young patients have poor adaptability to dietary adjustments and low cooperation; the ketogenic diet imposes strict requirements on dietary structure and has a long treatment course (usually measured in years), which is difficult for ordinary families to maintain—ultimately leading to suboptimal therapeutic outcomes. In addition, the ketogenic diet has an undesirable taste, which reduces patients’ quality of life. Over the long term, this exerts a significant negative impact on patients’ mental health. Based on the numerous advantages, shortcomings, and mechanism of action of the ketogenic diet, we aim to colonize engineered bacteria capable of efficiently synthesizing β-hydroxybutyrate (BHB) in the human body. These bacteria will enable the efficient and stable production of ketone bodies in vivo while bypassing the hepatic ketogenesis process to avoid side effects, thereby achieving the goal of treating epilepsy.

Wet Lab

Synthesis of BHB

By referring to various existing BHB synthesis pathways, in our project, we introduced the genes encoding β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) from Ralstonia eutropha, and propionyl-CoA transferase (pct) from Megasphaera elsdenii into Escherichia coli (E. coli), successfully constructing a BHB biosynthetic pathway. This pathway couples the catalytic reaction of PCT with the regeneration of acetyl-CoA (the starting material for BHB synthesis) and enables the excretion of BHB. Considering the reaction equilibrium of PCT, since acetic acid acts as an acceptor for CoA and is abundant in the human intestinal tract, the PCT-catalyzed production of BHB is expected to utilize intestinal acetic acid to promote BHB synthesis while reducing the metabolic burden on the engineered bacteria.

We constructed two plasmids. In addition to the genes encoding the three aforementioned enzymes, these plasmids were also linked to the gene encoding the BHB transporter protein RplO. Specifically, rplO and phaA were inserted into plasmid pRSFDuet-1, while phaB and pct were inserted into plasmid pET-Duet-1.

We introduced the recombinant plasmids into the engineered strain E. coli Nissle 1917, which then metabolized glucose as a substrate. Through the reaction between BHB and NAD⁺, a characteristic color was ultimately produced, verifying the BHB-producing metabolic pathway. We also designed experiments to investigate the relationship between the population size of the engineered bacterial community and the product yield, aiming to provide better guidance for product development.

Fig 10. The Synthetic Pathway of BHB

Regulation of BHB

When patients undergo ketogenic diet therapy, the clinical reference range for blood BHB concentration is 1.1–4.9 mmol/L. Therefore, we need to add a regulatory pathway. Initially, we considered regulating BHB using siRNA-mediated gene silencing technology and enzyme substrate concentration-responsive promoters. However, due to the lack of components responsive to the relevant substrates, we ultimately opted for a quorum sensing-based dynamic regulatory circuit, which offers the highest level of biosafety. The constitutive synthase LuxI produces signaling molecules. At high bacterial density, the accumulated signaling molecules are recruited by the transcription factor LuxR, which then binds to the Plux promoter and induces the expression of the bacterial toxin ccdB, thereby reducing the bacterial density. We verified the relationship between bacterial population density and BHB production, and based on this, selected a Plux promoter with an appropriate sensitivity to control the bacterial density within the required range.

Fig 11. Expression and Regulation of BHB

Suicide Pathway

To ensure that engineered E. coli colonizing the intestine initiates suicide after leakage into the extraintestinal environment, and survives in the non-intestinal environment before taking the drug, we designed the following oxygen concentration suicide system with additional pre-dose precaution. The system consists of three modules : PhlF repressor protein controlled by PfdhF promoter, ssrA-tag modified CI repressor controlled by PhlF promoter, operon formed by PVHb promoter and OR operator, and ccdB toxic protein controlled by it. The Tet R family repressor regulatory promoter PhlF from B.subtilis promotes the expression of Cl repressor with ssrA-tag, specifically binds to the OR operator, and acts as an precaution to prevent the activation of the microaerobic promoter PVHb before medication and initiates the suicide mechanism.After administration, the hypoxia promoter PfdhF is activated by the anaerobic environment in the intestine, and PhlF repressor protein is expressed and specifically binds to PhlF and inhibits its function, that is, Cl repressor is expressed. The ssrA-tag ensures that once the induction signal stops the synthesis of CI protein, any pre-existing CI molecules in the cell will be rapidly degraded, relieving its inhibition of the micro-oxygen promoter PVHb. Since then, once the engineered bacteria leaked from the anaerobic environment of the intestine and came into contact with a higher oxygen concentration environment, PVHb would activate ccdB and cause the engineered bacteria to kill themselves.

Fig 12. Suicide Mechanism of Hydrogel

Hydrogel System

To ensure that the engineered bacteria colonize the small intestine of the digestive tract as planned, while avoiding complex interactions between the engineered bacteria and the intestinal flora as well as biosafety issues such as severe gene leakage caused by horizontal transfer of genetic material, we innovatively propose a solution of encapsulating the engineered bacteria in hydrogel. This allows the "drug" (encapsulated bacteria) to physically adhere directly to the intestinal tract without the need to introduce additional elements into the bacterial cells for expressing adhesive substances; it also physically isolates the engineered bacteria from the intestinal flora, ensuring the stable presence of the bacterial cells in the digestive tract. By reviewing relevant literature, we have designed a hydrogel mainly composed of sodium alginate (SA) and polydopamine (PDA), following a three-layer structure design concept: The outer layer is a pH-responsive layer, which degrades upon reaching the weakly alkaline environment of the intestine, exposing the middle layer. The middle layer is a coating made of polydopamine, sodium alginate, and polyacrylamide. It adheres to and colonizes the intestine to synthesize and secrete β-hydroxybutyrate (BHB). Moreover, the pore size of the middle layer is too small to allow the passage of macromolecules such as DNA, preventing horizontal genetic material transfer with intestinal microorganisms and enhancing safety. The inner layer is a nutrient core formed by mixing alginate with bacterial suspension, providing the nutrients required for the survival of the engineered bacteria.

Fig13. Intelligent Hydrogel

Overall, after the drug made of bacteria encapsulated in hydrogel is ingested, it reaches the alkaline environment of the small intestine and adheres there. The engineered bacteria produce β-hydroxybutyrate (BHB) according to the established metabolic pathway; the BHB is then effluxed out (of the bacteria) and absorbed into the bloodstream by the small intestinal epithelium. Through blood circulation, BHB is transported to the brain, and with the assistance of carrier proteins, it crosses the blood-brain barrier to reach the lesion site, ultimately exerting its effect in treating epilepsy.

Fig 14. Drug Entry into the Body and Its Action

An Idea

Following the teacher’s advice and through literature research, we have designed an alternative BHB delivery method: hydrogel microneedles. The BHB delivery system based on hydrogel microneedles provides an innovative, use biocompatible polymer hydrogel materials (gelatin, polyethylene glycol, etc.) to construct a microneedle array, and BHB can be pre-loaded in the reservoir of the needle. When the microneedle patch is applied to the skin, the needle body penetrates the stratum corneum and rapidly swells after contacting the tissue fluid to form a continuous microchannel, which promotes the continuous release of BHB and penetrates into the dermal microcirculation, and then enters the systemic circulation to exert antiepileptic effects. This system has multiple advantages: First, painless and minimally invasive, improve patient compliance, suitable for long-term administration; second, the sustained release of BHB can be achieved by adjusting the hydrogel crosslinking degree. The third is to avoid the first-pass effect and improve the efficiency of delivery; fourth, it is convenient to use, and patients can apply it themselves, which is suitable for family and community medical scenes. Due to time constraints, our team has not yet conducted in-depth design and experiments on the hydrogel microneedle concept. In the follow-up supplementary work of the team, we will continue to explore this scheme.

Fig 15. Hydrogel Microneedle Drug Delivery

Dry Lab

Overview

Unlike the wet lab section which attempts to make synthetic biology-based contributions to epilepsy treatment, the dry lab section is dedicated to providing support in the prevention and monitoring of epilepsy. The dry experiment section is mainly composed of three parts: hardware, software, and model. Using basic electrophysiological signals as tools, we have successfully built an epilepsy monitoring and early warning platform based on electroencephalogram (EEG) signals through the design of signal extraction, cleaning and delivery, and model training. To a certain extent, it provides feedback support for the wet experiment content, with the aim of offering different perspectives for the prevention and treatment of epilepsy from various angles.

Model

When epilepsy occurs, the EEG shows three distinct waveforms that are significantly different from the normal state: spike waves, slow waves, and spike-slow complex waves. Clinical diagnosis mainly relies on the rich experience of doctors. To provide a reliable and accurate prediction model for both doctors and patients, we used machine learning modeling to extract the temporal and frequency features of EEG signals during epileptic seizures and conduct model learning. After a period of training, we achieved a relatively high accuracy of 80.88%. The training and establishment of this model laid a solid foundation for dry experiments.

Fig 16. Model Training Results

In addition, β-hydroxybutyric acid (BHB) is a molecule that plays a role in the ketogenic diet therapy for epilepsy. It is reasonable to pay attention to its metabolic process. To correct the differences in the metabolic process caused by different physiological characteristics among individuals, we built a population pharmacokinetic model of BHB. After inputting various physiological characteristic indicators, the model will provide the predicted BHB metabolic curve under the given conditions and return the recommended dosage based on this, serving as a reference in clinical scenarios.

Software

The EEG signals extracted from the electroencephalogram (EEG) devices often contain various types of noise and artifacts, which prevent people from making good use of them and obtaining the expected information. Therefore, it is particularly important to develop a set of software for cleaning and processing EEG information as well as visualizing the information. We have completed the development and construction of two software programs, EEG QuickLab and ES Detection. The former has made great efforts to achieve rapid processing of raw EEG signals, laying the foundation for the realization of the latter's tasks. The latter uses the clear data delivered by the former to predict models, calculates the frequency of abnormal EEGs within a certain period of time, and provides an epilepsy risk index as an important assessment for the risk of seizure. In this way, the two have achieved a leap from raw EEG signals to information visualization and play an extremely important role in the epilepsy monitoring and early warning platform based on EEG signals

Fig 17. EEG: QuickLab Interface

Hardware

How to simply and quickly extract EEG signals is a matter worth considering. We have developed a full-brain EEG extraction device, using the OpenBCI circuit board and the corresponding OpenBCI GUI for signal extraction and display. Considering the exposed features of the head-mounted EEG extraction device, epilepsy patients are easily affected. To avoid this social embarrassment, we further designed an ear-behind EEG extraction device, cEEGrid, which arranges an eight-electrode flexible array behind the ear to extract EEG signals. We have replaced the cumbersome EEG cap device with a lightweight device similar to headphones, achieving our goal.

Fig 18. Schematic Diagram of cEEGrid

Positive Impacts and Objectives

Traditional ketogenic diets cause many side effects—such as cardiovascular and cerebrovascular diseases, and poor development in adolescents—due to altered dietary structures. Our solution allows epilepsy patients to maintain a normal diet, avoiding these side effects while still achieving an epileptic therapeutic effect. After taking our engineered bacteria capsules, the body can produce BHB on its own. Patients can choose their preferred foods without being restricted by the dietary requirements of traditional ketogenic diets. Long-term efficacy is achieved with the engineered bacteria, eliminating the need for frequent medication intake and effectively improving patients’ quality of life. Our solution involves colonizing engineered bacteria in the intestines to produce ketone bodies endogenously. This significantly reduces medication frequency, prevents issues like missed or excessive doses, and maintains a consistent therapeutic effect. Meanwhile, the absence of dietary restrictions makes patients more psychologically receptive to treatment, greatly improving adherence. Epilepsy patients can wear our designed concealed behind-the-ear device. This device collects EEG signals, which are then received via Bluetooth and processed by software. It enables real-time monitoring of patients’ conditions, assesses seizure risk, and provides early warnings before a potential seizure—alerting family members or caregivers to prevent accidents. During the project, we carried out a series of epilepsy-related popular science activities targeting different age groups. For children, we created picture books; for adolescents, we provided training on first aid for epilepsy patients; and in schools, we organized activities to eliminate prejudices against epilepsy patients. Through these efforts, we aim to convey the message of caring for epilepsy patients to society.

Limitations and Potential Risks

lack support from in vivo efficacy and safety data. As animal models or preclinical studies have not yet been conducted, key issues—such as the colonization behavior of engineered bacteria in the living intestinal tract, the transmembrane transport efficiency of BHB, the immunogenicity of long-term use, and its potential impact on intestinal flora—all require further experimental verification. From a translational perspective, the innovative therapy of oral live genetically engineered bacteria will face strict regulatory review. The public’s psychological acceptance of "genetically modified bacteria" is also a barrier that needs to be overcome in the future. Additionally, if this therapy is eventually successfully launched, attention should be paid to its potential high price as an innovative drug—to avoid exacerbating social inequality in epilepsy treatment resources.

Next Steps

We aim to develop a new, safe, and effective treatment for epilepsy. In the future, we plan to conduct further research and add experimental and quality inspection steps—including animal model experiments, genetic stability testing, pharmacological and pathological studies, and clinical safety trials—to ensure the successful market launch of our product. For our hardware, we intend to achieve the practical application of our behind-the-ear device through reliability testing, biocompatibility tests, electrical safety certifications (e.g., CE, FCC), and software validation, bringing new hope to the numerous epilepsy patients worldwide. Going forward, we will continue to monitor advancements in epilepsy treatment while carrying out public outreach activities. Through short videos, comics, live broadcasts, and other formats, we will demystify epilepsy, introduce cutting-edge treatments, share patients’ stories, and dispel "demonized" prejudices, striving to increase global inclusivity for epilepsy patients. During the development of our project, we discovered that ketone bodies play multiple roles in the human body and can alleviate many diseases—especially neurological disorders—such as obesity, malignant tumors, and Parkinson’s disease. Based on this, we will continue to explore other application scenarios for our project. In terms of neurological diseases, ketone bodies exhibit excellent neuroprotective and regulatory effects; they provide neuroprotection and symptom relief in Alzheimer’s disease, Parkinson’s disease, migraine, traumatic brain injury, and amyotrophic lateral sclerosis (ALS), making them a promising option for treating chronic neurological disorders. In metabolic diseases, as key participants in energy metabolism, ketone bodies have shown potential in treating type 2 diabetes, managing obesity, and addressing non-alcoholic fatty liver disease, and we are exploring how to use our platform to precisely regulate systemic energy metabolism, enabling a novel, minimally invasive approach for long-term disease management. From start to finish, we firmly believe that synthetic biology is not just a technology, but a new paradigm for problem-solving. Starting with our "Epilepsy Shield," we hope to use synthetic biology methods to address a variety of human health challenges.

Wishes

Based on our current achievements, we hope this innovative therapy can bring some positive changes to epilepsy treatment worldwide. If subsequent verification goes smoothly, this synthetic biology-based therapy may help patients alleviate some of the side effects and dietary restrictions associated with traditional ketogenic diets, offering a potential option to explore for improving patients’ quality of life. We sincerely hope that this project can gradually advance toward application in the future and provide a new reference for treatment approaches to the approximately 50 million epilepsy patients across the globe. At the same time, we believe that if this comprehensive scheme—integrating synthetic biology and intelligent monitoring systems—can be continuously refined, it is expected to provide some support for the transition of epilepsy treatment from "symptom control" to "precision prevention and personalized treatment." By continuously optimizing the safety and stability of engineered bacteria and gradually improving the "monitoring-treatment" intelligent closed loop, we hope to offer a preliminary practical reference on safety and ethics for our peers in their exploration of related fields. Enabling technological innovation to better serve human health is the original aspiration we have always upheld. We hope that the "Epilepsy Shield" can play a certain role in safeguarding patients’ health in the future, and that the preliminary practices of this project in biosafety and ethical standards can accumulate some experience for the safe application of synthetic biology in the medical field. We hope to use our efforts to create an inclusive environment for epilepsy patients, not only within the iGEM community but also around the world.