In the process of human practice, we must critically examine our projects, achieve continuous optimization of projects, and think deeply about the impact of our projects on the world. We must continue to improve and optimize our projects with reflection and insight. During this social practice, we not only interviewed experts in related fields, but also gained an in-depth understanding of the feelings and needs of patients' families. These valuable exchanges and experiences make our projects richer and deeper.

Phenylketonuria, also known as phenylalanine hydroxylase deficiency, is a genetic disease caused by abnormal metabolism or reduced activity of phenylalanine. The disease is not easily detectable in its early stages and usually appears in the newborn period. If not diagnosed and treated promptly, children may show delays in intellectual and physical development as they grow. In severe cases, patients may even suffer intellectual disability. Therefore, it is crucial to provide timely and accurate treatment as well as scientific care to patients with phenylketonuria to ensure that they can grow up healthily.
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When our classmate was browsing the WeChat public account, an article titled "Angels Who Can't Eat Fireworks from Earth" aroused his strong interest. After further reading, we learned that this is actually popular science content about phenylketonuria.
Patients with phenylketonuria seem to be born with a rejection of the chicken, fish, meat, and eggs that ordinary people like. These foods are like fatal "poisons" to them. They have to endure the suffering of "what they want but can't get it" and can only eat specially prepared food for a long time. Therefore, they are vividly described as "angels who do not eat fireworks from the world", which is what we often call patients with phenylketonuria (PKU).
However, the price of these specially prepared foods is much higher than that of ordinary food, placing a heavy financial burden on their families. What's even more serious is that they must continue to strictly control their diet. A little carelessness may lead to severe intellectual disability, epilepsy and other neurological problems.
Current treatments are not perfect, have side effects, are expensive, and have varying effects from person to person. Because of these challenges, we were deeply inspired and decided to delve into a more effective and humane treatment plan, and took this as a topic to participate in this year's igem competition.

Contact a Phenylketonuria Doctor for an Interview

After in-depth discussions with doctors who treat phenylketonuria, we have a more detailed understanding of current treatments.
The first treatment strategy is to supplement the lack of BH4 by taking BH4 coenzyme drugs, thereby increasing the activity of PAH and further assisting the degradation of Phe. The second strategy is to directly break down Phe by ingesting PAL drugs. This is an innovative treatment path designed to more directly address the accumulation of Phe. While undergoing the above treatments, patients also need to follow a low-Phe diet and directly supplement amino acids.
After in-depth understanding, we found that the two most popular treatments for patients are: KUVAN sapropterin dihydrochloride and Palynziq.
KUVAN sapropterin dihydrochloride is the only oral drug combined with dietary adjustment. Its mechanism of action is to absorb BH4 coenzyme, thereby increasing the activity of PAH and helping to degrade Phe. Palynziq is currently the only injectable drug that uses PAL to replace Phe enzyme. According to the US FDA approval, its maximum allowable dose is 60mg.

However, after communicating with the families of children with phenylketonuria, we learned that Palynziq has a series of adverse reactions, including injection site reactions, joint pain, hypersensitivity reactions, headaches, and systemic skin irritation that lasts for two weeks or longer. reaction. Regrettably, children have to endure these adverse reactions at least 20% of the time during each treatment cycle.
We are eager to find ways to address the adverse reactions and side effects associated with these drugs and provide better treatment options for patients with phenylketonuria.

Team discussion

After our offline discussions, we explored a new treatment idea: using probiotics to absorb and decompose Phe in the intestines, thereby reducing the patient's intestinal absorption pressure on Phe. This method aims to start from the intestinal environment and create a more beneficial intestinal microecological balance for patients, thereby further helping patients alleviate their symptoms.
We focus on bioengineering technologies, especially the use of E. coli as a microbial chassis for gene editing. We chose Escherichia coli because of its low cost and mature gene editing technology.

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Although E coli BL21 (DE3) is often used for this type of project, after reviewing our data, we decided to use E coli Nissle 1917. This E. coli is endotoxin-free, ensuring the health and safety of those taking it. To this end, we modified E. coli and introduced phep, an enzyme responsible for phe transport.

Interview with Dr. Zhou

During the experiment, we noticed that the absorption effect of phe by E. coli was not as expected. To find out what the problem is, we asked Dr. Zhou, who specializes in the field of biology.

Our initial design plan was to transfer part of the phenylpyruvic acid in the patient's body to the bacteria. However, due to the limited capacity of the bacteria, when the amount of phe produced in the body exceeds the absorption capacity of the bacteria, the Phe concentration in the bacteria will be saturated and may Lead to excessive accumulation of Phe in the body.
In order to solve this problem, we reconsidered the plan and planned to introduce a degradation step to enable E. coli to effectively excrete Phe from the body, thus ensuring that the bacteria can continue to absorb and process Phe.

Ask a Toddler Therapist

After speaking with early childhood therapists, we narrowed down and eliminated several potential transforming substances. Our team members discovered that trans-cinnamic acid is a common metabolite of coliform bacteria. This substance can be converted into hippuric acid in the liver and eventually excreted in the urine. According to Guts' research paper, high concentrations of hippurate are considered indicators of metabolic health. Especially in obesity caused by a high-fat diet, hippurate can help improve body metabolism.
This finding was confirmed and affirmed by early childhood therapists. On this basis, we decided to use trans-cinnamic acid as the main metabolite of E. coli.
Multiple studies have shown that high concentrations of hippurate are positively associated with metabolic health, both in humans and animals. Hippurate plays a role in improving metabolism in obesity caused by high-fat diet. Combining these findings, we selected trans-cinnamic acid as a key metabolite of E. coli and expected to promote the metabolic health of patients through this pathway.

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We consider adding a step of degradation to convert the absorbed E. coli into another substance (trans-cinnamic acid) that the human body can metabolize normally, and excrete it to be absorbed and metabolized by the human body.

Control of the amount of E. coli metabolites

Protein is widely found in nature, but its content is not always high. For example, in eggs, dairy and animal-derived proteins, the content is usually 5-6%; in plant proteins, the content is only 1%; and the content of single-cell proteins is between 3% and 5%.

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According to our data review, the recommended daily protein intake for adolescents aged 14 to 18 is 52 grams, which is equivalent to 0.85 grams per kilogram of body weight per day. In other words, the recommended intake of protein is 0.39 grams per pound of body weight. From our calculations, we learned that the content of trans-cinnamic acid converted by E. coli is approximately 2.5 grams, and this amount will not have any impact on the patient's health.

Product conversion

After consulting with lawyers and conducting a biosafety assessment, we learned that there are many challenges to using genetically modified microorganisms for human therapeutics in China. In addition, most patients and their families have psychological barriers to directly consuming E. coli, and they prefer to avoid direct contact between the human body and these microorganisms. Therefore, we optimized the original in vivo treatment plan and converted it to an in vitro preparation method.
We retained the transporter gene design and the main structure of the PAL enzyme in the original scheme, but made necessary modifications to the promoter part of the PAL gene sequence. This aims to ensure the biosafety of microorganisms while meeting therapeutic needs.
We will manufacture low-cost special dietary supplements for patients with phenylketonuria.

In vitro product production safety standards

When communicating with lawyers, we were directed to GB29921-2013 National Food Safety Standard - Limits of Pathogenic Bacteria in Food. This standard clarifies the limits, detection methods and requirements of pathogenic bacteria in prepackaged foods. The pathogenic bacteria involved include Salmonella, Listeria, Staphylococcus aureus, Escherichia coli O157:H7 and Vibrio parahaemolyticus. In order to ensure food safety for consumers, we will emphasize this standard when we contact manufacturers and require them to strictly follow the limits on pathogenic bacteria.

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Special meal selections

In interviews with the company, we learned that treatment for phenylketonuria needs to begin in infancy. Therefore, we plan to use genetically modified E. coli in the production of milk powder. Using this technology, we are able to create a specially formulated milk powder that does not contain phenylalanine, ensuring it is safe for children to ingest. In addition, the preparation and culture process of E. coli is relatively simple, which can not only effectively reduce production costs, but also relieve financial pressure on families and benefit more patients.

Charity organization

In view of the generally limited financial conditions of many families with phenylketonuria, we feel deeply responsible and missionary and decided to add a special chapter in the business plan - "Charitable Foundation Plan". The plan aims to set up a special fund to provide financial assistance to families who need but cannot afford specialized milk powder through fundraising and donations. We hope that through this plan, we can not only reduce the financial pressure on families with children, but also arouse concern and support from all walks of life for families with phenylketonuria. We firmly believe that every small act of kindness helps create a more inclusive and caring society, so that every child with disease can receive timely and appropriate treatment.