During our brainstorming process, we realized the mighty potency of synthetic biology in medical treatments, especially in the therapy development of inflammatory bowel disease (IBD), one of the hardest-to-cure conditions in the world. Thanks to the well-understood genetics and its efficient heterologous protein expression capacity, Saccharomyces cerevisiae appears to be a promising chassis organism for developing biological therapies for IBD. Additionally, various synthetic biology components enable us to design multiple control systems, achieving precise and comprehensive control over the treatment. Viewing IBD treatments from a brand-new aspect, we found that biological therapy obtains tremendous advantages not found in traditional chemical drugs. As a result, we have chosen IBD therapy as our project focus of this year.
Comprising of Ulcerative Colitis (UC) and Crohn's Disease (CD), IBD is a chronic, incurable disease affecting people of all ages worldwide. According to the Global Burden of Disease (GBD), there was a notable increase of 175,904 individuals diagnosed with IBD from 1990 to 2021. Over 1 million residents in the USA and 2.5 million in Europe are estimated to have IBD, with substantial costs for health care. IBD patients normally suffer from abdominal pain and diarrhea, combined with intermittent fever and various extraintestinal symptoms such as arthritis. Besides, IBD is well-known for its complex and unclear pathogenesis, which also explains why no common and satisfying therapy exists.
In our research effort, we discovered that abnormal immune response triggered by the intestinal flora is strongly related to the disease. Meanwhile, recent studies have revealed that lactate can stabilize HIF-1α, which further limits mtROS production and finally reduces CNS autoimmunity. Because of its capability of relieving the inflammation in the intestine, we consider lactate as a promising micromolecular to treat IBD.
As for the method of administration, we have turned our attention to the traditional Chinese medicine musk. Musk is renowned for its use in treating various conditions such as stroke, coma, neurasthenia, convulsions, and heart diseases in China. Luckily, recent studies have found the receptor of muscone, the primary active components of musk, which inspired us about the idea of inhalational administration. For a long time, IBD patients have been complaining about traditional oral administration which easily leads to nausea, vomiting or even inflammatory response. In an attempt to balance both efficacy and comfortability, we march forward to developing inhalational administration using muscone.
Current treatments of IBD include surgical interventions, pharmacological therapies and nutritional therapies. However, these conventional IBD treatment methods still exhibit several limitations.
Surgical interventions play an important role in the treatment of IBD, especially for patients who do not respond to medical therapy or who have complications. Surgical interventions are very effective, but because of the widespread use of ostomy techniques, they can have a significant negative impact on the patient's daily life as well as the injury.
Pharmacological therapy is the core of IBD treatment, mainly including the following categories: aminosalicylic acid, glucocorticoids, immunosuppressants, biologics, antibiotics, probiotics, anticholinergics, etc. These drugs are usually immunosuppressive and increase the risk of infection with long-term use; Special targeted drugs have the disadvantage of being expensive.
Nutritional therapy plays an important role in the remission and prognosis of IBD. However, at present, nutritional therapy is faced with problems such as slow effect, long course of treatment, and insufficient targeting, which will affect the quality of life of patients.
| Treatment options | Cost | Performance | Indications | Side effects | Drawback |
|---|---|---|---|---|---|
| Surgery | High | Best | Refractory to medical treatment, serious complications | Surgical risks, postoperative infection, anastomotic leakage | Invasive, may require long-term rehabilitation, and is irreversible |
| Drug | Medium to high | Good | Mild, moderate, and severe, with no indication for surgery | Infection, liver and kidney damage, drug allergy | Long-term, severe side effects, individual differences in efficacy |
| Nutritional therapy | Low to medium | Average | All, especially the malnourished | Electrolyte imbalance, nutritional formula intolerance | Poor effects, require professional nutritional guidance |
In recognition of the limitations of current IBD therapy, we embarked on a journey to harness the power of synthetic biology in developing a biological therapy, which we named “MusCure” as the abbreviation of “Muscone to Cure”. Our primary objective was to engineer yeast capable of specifically colonizing at the inflammatory sites of the intestine and delivering lactate in response to the signals of muscone.
Based on the published paper and our patient interviews, oral administration has many drawbacks. For instance, parts of IBD patients have difficulty in swallowing the pills, and given the vulnerable intestine of the patients, oral administration can cause further nausea or vomiting. To address the shortcomings of oral administration, MusCure offers an “APE” solution: “Affordable, Potential and Efficiency IBD treatment”. The following sections will elucidate how our design accomplishes these goals.
Saccharomyces cerevisiae is a premier chassis organism for our project. As a kind of common yeast found in human gut, Saccharomyces cerevisiae is considered safe for human and has been extensively studied for its potential application in synthetic biology. As a eukaryotic organism, it possesses more complex and precise mechanisms for gene expression regulation, and various modification approaches also enhance its ability to secrete target proteins. Of note, it has been employed to prevent and treat various diarrheal disorders due to its capability to reshape the balance of gut flora. We believe that Saccharomyces cerevisiae is the best chassis organism to achieve our goal.
We hope to provide a non-invasive and gentle drug delivery method for IBD patients. Therefore, we designed muscone as a molecular switch for the drug delivery engineering strain. We transferred the muscone receptor from mouse olfactory epithelial cells into Saccharomyces cerevisiae. By modifying the mating pathway of Saccharomyces cerevisiae, we enabled the muscone receptor to function as a signal switch in the yeast.
We assisted the muscone receptor in functioning as a signal switch in Saccharomyces cerevisiae through the G protein-coupled pathway by introducing a Gα protein modified with 5 amino acids at its C-terminus. We also linked the lactate dehydrogenase gene downstream of the promoter in the mating pathway. In the presence of muscone molecules, the molecular switch is activated, and the expression of lactate dehydrogenase is initiated through the mating pathway of the G protein-coupled Saccharomyces cerevisiae.
We altered the anaerobic metabolic pathway of Saccharomyces cerevisiae by introducing lactate dehydrogenase, enabling it to produce D-lactate and secrete it into the surrounding environment for the purpose of inhibiting the abnormal activation of immune cells in the intestine, thereby alleviating the symptoms of IBD patients.
To eliminate the signal interference from the natural mating process of Saccharomyces cerevisiae, we knocked out the original mating receptor STE2 in the yeast. Through experimentation, we confirmed that the knockout reduced the background noise signals in the wild-type yeast, thereby enhancing the stability of the system. In the experiment, we adopted a strategy of separately testing the upstream and downstream components of the system before integrating and validating them. First, we used the GFP reporter gene instead of lactate dehydrogenase to test whether the muscone molecular switch introduced into Saccharomyces cerevisiae could function properly. Meanwhile, we used the galactose promoter to express lactate dehydrogenase, verifying whether lactate dehydrogenase could alter the anaerobic metabolic pathway of Saccharomyces cerevisiae and successfully secrete D-lactate. After that, we synthesized the complete biological system and validated that under the muscone signal, the yeast could synthesize and secrete D-lactate.
For more details, see the Therapy system.
To enable our engineered Saccharomyces cerevisiae to specifically function at the small intestinal lesions of IBD patients, we have designed the colonization system. This system consists of two main components: the tetrathionate sensor TtrSR and the adhesion protein Als3. TtrSR can detect the chemical signals of extracellular IBD marker tetrathionate and promote the expression of downstream genes in the signaling pathway. Als3 is a cell surface protein from Candida albicans that acts as an adhesin, mediating adhesion to epithelial cells, endothelial cells, and extracellular matrix proteins. We have chosen Saccharomyces cerevisiae as the chassis organism for our project. By constructing the TtrSR system in Saccharomyces cerevisiae and expressing Als3 protein downstream, we aim to achieve specific colonization at the small intestinal lesions of IBD patients, which enables our therapy systems to work better and more efficiently.
For more details, see the Colonization system.
In the dry lab, the prediction was made by modeling. Our model is divided into four interconnected parts, representing the inhalation of muscone, its binding to receptors, intracellular signal transduction and lactic acid secretion triggered by receptor activation, and the absorption of lactic acid. These models provide a comprehensive understanding of the project and yield valuable computational results. In addition, we have designed an electronic sachets with the implementation of therapy. We use emulsification via ultrasonication to make muscone evaporate from portable sachet bag and integrate traditional Chinese incense culture elements. For detailed information, please refer to Model or Hardware.
As a biological treatment we are developing, its safety has always been at the top priority for us. To ensure our engineered yeast can’t survive in the outside the gut, we introduced a suicide system using specific intestine marker, bile acid. In the intestine, bile acid binds to its receptor, which further activates the expression of CI protein. CI protein can inhibit the expression of suicide gene MazF. Once the yeast leaves intestine, lack of bile acid leads to successful expression of MazF, killing the engineered yeast. By introducing this system, we ensure that our yeast can only colonize and thrive in the intestine. Besides, nutritional-deficiency also provides a convenient and efficient approach for us to ensure biosafety. Our engineered yeast is uracil and histidine auxotrophic, further ensuring its safety. For detailed information, please refer to Safety.
Due to time and other constraints, we may not be able to achieve a more comprehensive design and analysis of MusCure. However, we hope to make further efforts in the following aspects to make this project more complete:
Traditional fermentation engineering uses Pichia pastoris for fermentation. When methanol is used as a carbon source, the expression of aldehyde oxidase (AOX) is greatly increased, and traditional fermentation engineering takes advantage of this characteristic to make the yeast express target genes located near the AOX gene. However, methanol has certain biological toxicity. Muscone itself is non-toxic and harmless, and as a water-soluble molecule, its recovery and reuse are relatively simple. We are exploring a system that uses muscone as a molecular switch, hoping to apply it to yeast fermentation engineering after further research.
In addition to lactate, we also plan to try some other small molecule drugs as target proteins for our engineered bacteria, further exploring the potential of MusCure in treating IBD and other diseases.
As for our models, different approaches can be applied to further enhance their performances. We are gonging to apply various and more comprehensive algorithms in our models to achieve more precise prediction.
We have completed preliminary design of our electronic sachet. To further improve its practicability, more complicated algorithms and more precise controlling elements can be applied. Also, more Chinese traditional culture elements can be integrated in order to optimize its appearance.
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