Butyric acid-producing engineered yeast as an adjunctive treatment for Alzheimer's disease: Core Protocol

I. Overview

We have completed the preliminary functional verification of butyric acid-producing engineered yeast in the laboratory (in vitro inhibition of neuroinflammation, cell model tests, plasmid transformation tests, etc.), and the ultimate goal is to develop an innovative product to assist in the treatment of Alzheimer's disease (AD). Through literature review and clinical needs research, it was found that the current AD treatment has the pain points of "single target, obvious side effects, and lack of early intervention" - traditional drugs mostly directly act on brain targets, with a complex medication process and causing considerable pain to patients.

We focus on the pathogenesis of the same disease in AD patients, which is "increased APOE4 expression → release of pro-inflammatory factors → activation of microglia → intensified neuroinflammation → intensified neuronal damage → memory and cognitive impairment", and transport butyric acid synthesized by engineered yeast in the intestine to the brain through the gut-brain axis. The inhibitory effect of butyric acid on neuroinflammation can assist in improving the cognitive function of AD patients and fill the clinical gap of early intervention.

It is worth noting that although we used model Saccharomyces cerevisiae (INVSc1) for proof-of-concept and functional testing in the laboratory stage, the transformation from the laboratory to the clinical stage requires the selection of more suitable chassis strains. Moreover, considering species homology, the ease of operation, and the current production on the market, Saccharomyces boulardii is the best choice. Saccharomyces boulardii was discovered by the French microbiologist Henri Boulard in 1923 (Offei B. et al. 2019). Over the following 100 years, this yeast strain has been widely used in clinical practice for the treatment of various types of diarrhea and intestinal diseases, and has accumulated rich data on safety and efficacy. Through research, we have demonstrated that the core gene modules we designed - including the TMA sensing system, ROS monitoring system, butyric acid synthesis pathway and tetracycline-induced suicide system - have excellent transferability and can be efficiently transformed into Saccharomyces boulardii, making them the preferred choice for future clinical applications. This strategy not only ensures the efficiency of research and development but also guarantees the safety and regulatory feasibility of the final product. (For more details, please visit our Engineering webpage.)

II. Target Audience

(1) Current target audience

As an adjunctive therapeutic product for regulating the gut-brain axis, our yeast focuses on patients with mild to moderate AD - according to Yuan J. According to the team's research, 50.4% of all AD patients had mild cases and 30.3% had moderate cases, totaling approximately 80% (Yuan J. et al. 2021). For such patients, the rate of cognitive decline is relatively slow, and the disorder of intestinal flora is not yet severe. Engineered yeast can delay the progression of the disease and reduce the cost of subsequent care through early intervention.

Fig. 1. Pooled percentages of AD dementia clinical syndrome by disease severity among participants with AD dementia. Pooled percentages of AD dementia by disease severity(mild, moderate, and severe)in three time-windows were illustrated among participant. with AD dementia. The 95% confidence intervals were showed by error bars

(Picture resource: Yuan J, Maserejian N, Liu Y, Devine S, Gillis C, Massaro J, Au R. Severity Distribution of Alzheimer's Disease Dementia and Mild Cognitive Impairment in the Framingham Heart Study. J Alzheimers Dis. 2021;79(2):807-817. doi: 10.3233/JAD-200786. )

According to literature reports, in China, based on a 2020 national cross-sectional study, there are 9.83 million AD patients aged 60 and above (Jia L. et al. 2020), among which it is estimated that there are approximately 7 to 8 million cases of mild to moderate AD patients. Meanwhile, the population with mild cognitive impairment (MCI) is also the core target group for AD intervention treatment. Studies have shown that approximately 10-15% of MCI patients transform into AD each year (Mitchell AJ, Shiri-Feshki M. 2009). Our yeast can reduce the risk of transformation through intestinal regulation, filling the gap in the "prevent-treatment" transition.

(2) Future Target Audience

With the intensification of aging, AD is showing a trend of "younger onset" (the proportion of patients under 60 years old in China exceeds 20%), and it is often accompanied by underlying diseases such as diabetes and hypertension. Intestinal disorders are more obvious in such patients. In the future, the perception module of engineered yeast can be optimized to adapt to the intestinal characteristics of patients with AD underlying diseases. At the same time, the potential for assistance to other neurodegenerative diseases (such as Parkinson's disease) can be explored - all of which have varying degrees of gut-brain axis abnormalities. The anti-inflammatory properties of butyric acid may have broad-spectrum auxiliary value.

III. Safety Issues

1. The necessity of a suicide system

Although Saccharomyces boulardii has shown good overall safety in clinical application, literature reports indicate that there are still rare but significant risks in specific high-risk populations. The main risk factors include ICU hospitalization, total parenteral nutrition, central venous catheterization, and immunosuppressive status. Another systematic review covering 117 cases of Saccharomyces boulardii mycosis fungemia from 2005 to 2022 showed that 67.6% of the cases (73 cases) occurred during probiotic treatment, with an all-cause mortality rate of 36.1% (Vinayagamoorthy K. et al. 2023). Although the overall incidence of these adverse events is extremely low (accounting for 0.1-3.6% of all mycoemia) (Riquelme AJ. et al. 2003), the consequences can be serious for patients with compromised immune function.

Given that AD patients are mostly elderly people, often accompanied by other underlying diseases, and have relatively weakened immune functions, they theoretically belong to the potential high-risk group. More importantly, the engineered strains carry exogenous genes. Although the probability of horizontal gene transfer from Saccharomyces boulardii as a eukaryote to prokaryotic bacteria is extremely low, from the perspectives of biosafety and ethical prudence, we still need to establish a final safety guarantee mechanism.

Based on the above considerations, we have designed a tetracycline-induced user-controlled programmed cell death system for engineered yeast. Under normal conditions, this system maintains "zero leakage" expression through silencing sub-components, without affecting the therapeutic function of the engineered yeast. Once a patient experiences a suspected immune rejection reaction (such as fever, rash, or severe gastrointestinal discomfort) or the clinician decides to terminate the treatment for safety reasons, oral tetracycline drugs can be taken to rapidly activate the rtTA3 transcription factor, strongly activate the expression of the pro-apoptotic protein Bax, efficiently eliminate all engineered yeasts in the intestinal tract within 3 to 5 days, block the possible further development of immune responses, and at the same time prevent the long-term residence of engineered strains and potential ecological impacts. (For details, please refer to our Safety and Engineering webpages.)

2. Risk assessment

Given that our project utilized engineered yeast, although we have modified it and incorporated a suicide system to reduce certain risks, there are still potential risks to public health and the environment. First, there is the risk of host safety. Although Saccharomyces boulardii has good clinical safety, there is still a rare risk of mycosis in AD patients with compromised immune function. Since both TMA and ROS levels are influenced by physiological factors such as diet, exercise, and acute infection, engineered yeast may be wrongly activated under non-pathological conditions. In addition, the long-term colonization risk also needs to be considered. The continuous colonization of engineered yeast in the intestinal tract may lead to long-term changes in the intestinal microecology or be difficult to be completely cleared after the treatment ends.

At the metabolic level, the butyric acid synthesis pathway may be overly activated under certain extreme conditions, consuming an excessive amount of the intracellular coenzyme A pool, affecting the metabolic balance of yeast itself, and even leading to cell death. Meanwhile, complex genetic circuits may accumulate mutations during long-term passage, leading to changes in sensor sensitivity or failure of safety switches. Although the probability of gene transfer between eukaryotes and prokaryotes is extremely low, the long-term ecological impact of the release of exogenous gene fragments into the intestinal environment still needs to be evaluated.

In accordance with the requirements of China's Biosecurity Law (implemented in 2021 and revised in 2024), we have conducted a comprehensive risk assessment of engineered yeast, covering key links such as risk monitoring and early warning, risk investigation and assessment, and information sharing. Based on this, we have implemented a multi-level security transformation strategy:

In response to the several potential risks mentioned above, we have currently completed the design of some security mechanisms

To address the risk of unexpected activation, we adopted a dual-input "AND Gate" logic design - requiring both ROS and TMA to increase simultaneously to activate the system, reducing the false positive rate to the theoretical square level and effectively preventing false activation of the system.

In response to host safety and long-term colonization risks, we have designed a tetracycline-induced suicide system - users can clear over 99.9% of engineered yeast within 3-5 days by taking tetracycline orally, providing a final safe exit mechanism.

Considering the safety of the chassis strain, we used the FDA-approvedSaccharomyces boulardii - which has clinically proven safety and self-limiting characteristics and will naturally clear after the administration is stopped.

3. Future optimization directions

In view of the other potential risks that may still exist in the current system, such as the instability of the metabolism of engineered yeast and the possible failure of the suicide system, we have decided to further modify and optimize our system based on the actual situation such as the effect evaluation and social response after its future use

In response to the risk of metabolic flux loss of control, we plan to introduce A metabolic load sensor - utilizing the endogenous promoter of yeast that is sensitive to the CoA/Acetyl-CoA ratio, it automatically down-regulates the expression of key enzymes for butyric acid synthesis when the coenzyme A library is exhausted, protecting the metabolic homeostasis of engineered yeast.

In response to the potential failure of the suicide system, we envision the construction of a dual safety switch system - adding a backup suicide pathway based on the ubiquitin-proteasome pathway on the basis of the tetracycline-induced mechanism, reducing the probability of simultaneous failure of both systems to below 10^-6.

In response to the demand for genetic stability monitoring, we are considering designing a "Canary" early warning reporting system - inserting mCherry fluorescent proteins between key functional modules to promptly detect genomic rearrangement or deletion events by detecting the proportion of dual fluorescence signals.

IV. Administration Instructions and Usage Methods

1. Product form

Our engineered Saccharomyces boulardii live biological therapy will be provided in oral capsule form. Each capsule is wrapped in an enteric-coated capsule shell and contains a freeze-dried preserved engineered yeast strain to ensure its stability and activity at room temperature. This design aims to protect the engineered strain from smoothly passing through the gastric acid environment and effectively colonizing in the intestine to exert therapeutic effects.

Enteric-coated capsule technology has been applied in the field of probiotic delivery for over 30 years. Its core advantage lies in the use of ph-sensitive materials such as acrylic resin and hydroxypropyl methylcellulose phthalate, which remain intact in the acidic environment of the stomach (pH 1-3), while remaining intact in the neutral or weakly alkaline environment of the small intestine and colon (pH >6.5) and achieves targeted release (Asgari S. et al. 2020 & Yus C. et al. 2019).

Saccharomyces boulardii preparations have a clinical application history of over 20 years in China, widely used in the treatment of acute diarrhea, antibiotic-associated diarrhea and intestinal flora imbalance in adults and children, and their safety has been fully verified. Multiple clinical studies have confirmed that Saccharomyces boulardii has a significant effect on treating acute diarrhea in children, which can shorten the duration of diarrhea by an average of 1 day and reduce the length of hospital stay by 0.85 days. In Chinese clinical practice, Saccharomyces boulardii powder is used at a specification of 250mg per bag (containing no less than 1.3×10⁹ CFU of the powder). For adults, 2 bags are used twice a day each time, and for children over three years old, 1 bag is used twice a day each time (McFarland LV, Li T. 2025). It has shown good safety and tolerance.

From this perspective, the administration method of yeast powder plus capsules that we will adopt is already very mature and has been widely used in the market. It can not only ensure the precise release of the drug but also avoid losses.

2. Administration protocol

It is recommended that patients take 1 to 2 capsules daily with meals to facilitate the intestinal environment and promote yeast colonization after meals. It is recommended to take it continuously for 3 to 6 months. The specific course of treatment should be adjusted based on the professional assessment of the clinician and the individual response of the patient to achieve the best therapeutic effect.

3. Safety termination mechanism

If the patient or the clinician decides to terminate the treatment, the programmed cell death system built into the engineered yeast can be activated by oral tetracycline antibiotics (such as doxycycline,100-200mg/ day for 3-5 consecutive days). This user-controllable "suicide switch" can efficiently eliminate all engineered strains in the intestines within a short period of time, providing patients with proactive safety guarantees.

V. From Lab to Market

Our intelligent engineered yeast therapy aims to provide a new perspective for addressing core challenges in Alzheimer's disease treatment. Compared with traditional approaches, it is expected to demonstrate the following key advantages in the marketization process:

1. Potential Economic Advantages

Current AD monoclonal antibody drugs (such as aducanumab and lecanemab) cost over $26,000-$56,000 per patient annually (Hering H. et al. 2025). Our project is based on microbial fermentation engineering production. If successfully developed and large-scale manufacturing is achieved, the production cost is expected to be significantly lower than that of traditional biological drugs due to the scalability and efficiency of the fermentation process, which is expected to provide broader patient accessibility.

2. Potential Advantages in Compliance and Safety

Current monoclonal antibody therapy requires intravenous fluids every 2-4 weeks and has side effects such as amyloid-related imaging abnormalities (ARIA) and infusion reactions, which significantly affect patient compliance. Designed for oral administration, our product takes advantage of the local colonization and site-specific effects of the engineered probiotic yeast in the gastrointestinal tract, theoretically avoiding the risks associated with systemic exposure and invasive procedures, and could significantly enhance the convenience of administration and patient tolerance.

3. Potential advantages of therapeutic mechanisms

Different from existing drugs that target downstream pathological aggregates (such as amyloid plaques) or provide symptom relief, our product uses "sense-compute-perform" intelligent circuits to intervene in diseases from the perspective of regulating the intestinal microenvironment and restoring the function of the gut-brain axis (Loh JS. et al. 2024). The machinery is made for the upstream stage of the pathological cascade, specifically targeting APOE4-driven neuroinflammation, with the goal of not only relieving symptoms but also offering the potential to delay or even prevent the progression of neurodegeneration.

4. Precise Regulation Potential

The core innovation of this project is the dual-signal logic gating design. Engineered yeast can sense changes in disease-related signaling molecules (TMA and ROS) in the intestinal environment in real time and autonomtically regulate therapeutic output (butyrate production). This "on-demand production" model achieves a better balance between efficacy and safety by avoiding undertreatment and overtreatment, and the system inherently adapts to fluctuations in disease status.

In summary, this project explores an optimized treatment paradigm for AD across multiple dimensions—including route of administration, mechanism of action, production economics, and therapeutic precision—through synthetic biology approaches. In subsequent development stages, we are committed to transforming these potential advantages into demonstrated product competitiveness through rigorous scientific validation, technological refinement, and clinical evidence generation.

VI. Future Plans

1. Further optimization of engineered yeast

Due to the significant differences in each person's metabolic level, the probiotics we are currently designing may not be able to achieve broad-spectrum prevention and treatment. Therefore, we plan to introduce more complex genetic circuits in the future. For example, by detecting different concentrations of biomarker combinations, the engineered yeast will be able to self-detect and determine the severity of Alzheimer's disease. And release butyric acid in stages and doses to achieve more efficient and effective personalized treatment.

At present, our plan is to use the most widely promoted way of taking Saccharomyces boulardii - to make it into a powder for consumption. In the future, we plan to add a layer of "PH-responsive enteric-coated coating" on the outside of the yeast powder, allowing the yeast to remain intact in the highly acidic environment of the stomach (pH 1-3). It will only dissolve in the relatively neutral or weakly alkaline (pH > 7.0) environment of the colon, thus achieving precise release in the colon area

2. Patent application

We will also establish a comprehensive patent portfolio to ensure the intellectual property protection of our key innovations, including dual-input gene circuit design, optimized TAAR5-based TMA sensing module and butyrate synthesis pathway configuration. These patents will not only protect our technological progress, but also demonstrate the novelty and commercial feasibility of our methods to potential investors and partners.

3. Social development

In the next stage of application, we will conduct a thorough technical feasibility analysis to evaluate the preventive and therapeutic effects of engineered yeast in various situations. Specifically, since the onset age of Alzheimer's disease is mostly middle-aged and elderly people, we need to consider the impact of many other diseases on the amount of butyric acid produced by yeast. We will simulate the colonization of yeast and the yield of butyric acid under different physiological conditions, such as the influence of various diseases, to ensure that our technology can well adapt to different physiological and pathological conditions.

To ensure that our engineered yeast can be better applied in production, we plan to engage in dialogue with bioethicists, legal experts and regulatory authorities to jointly explore the ethical challenges that may be encountered in the clinical application of this live biopharmaceutical.

4. Sustainable production and global accessibility

To enable our engineered yeast therapy to benefit patients worldwide, we are planning to optimize the cost-effective manufacturing process to make the treatment price competitive compared with the existing high-cost therapies. Our goal is to make the production cost significantly lower than that of current monoclonal antibody treatment drugs, so that patients from different socioeconomic backgrounds can afford it and receive treatment. We also plan to establish partnerships with international health organizations, including the World Health Organization and regional disease control agencies, to obtain support from social institutions for our drugs and to facilitate regulatory approval and distribution in low - and middle-income countries. Finally, we plan to implement a technology transfer program to make local production capacity possible in partner countries, reduce reliance on centralized manufacturing, and lower costs through regional production networks. This multi-faceted approach ensures that our innovations not only serve affluent markets but also benefit underserved populations that bear a disproportionate burden of Alzheimer's disease.

Reference

Details

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