Integrated Human Practices

Dialogue with Glaucoma Friend Network (GFN)

Glaucoma patient organizations.

Glaucoma Friend Network (GFN) is a membership organization run by and for glaucoma patients. Their activities are of wide variety, including study sessions on glaucoma, the publication of newsletters, encouraging interactions among members, and raising public awareness of the significance of early detection of glaucoma.
Through the dialogue with GFN, we aimed to hear opinions from patients about the glaucoma detection and the importance of early detection.

Interview

The members of GFN showed strong interest in our activities. They not only provided us with valuable feedback on our project but also shared their concerns and the burdens they experience regarding their treatment as patients.
Firstly, as for the feedback on the glaucoma detection device for household use itself, concerns were raised about the possibility that users might mistakenly assume they are fine with a false negative result.
They shared with us that many patients, after being diagnosed with glaucoma, panic after searching on Google about glaucoma symptoms or become depressed due to the fear of going blind. This has even led some to quit their jobs or suffer from significant emotional stress. They emphasized the importance of telling that early detection can help slow the progression of the disease, to prevent such misunderstandings.
From these points, since glaucoma often presents no noticeable symptoms in the early stages, they suggested that a detection device we propose could potentially reduce psychological anxiety.
They also mentioned that the fact that glaucoma diagnosis needs an amount of time makes frequent hospital visits burdensome. Moreover, even after being diagnosed with glaucoma, patients often need to apply multiple types of eye drops, which can be quite tedious. Due to such burdens and the lack of obvious symptoms until the condition progresses, some people stop attending their appointments on their own, highlighting the potential of our device to raise awareness and create a sense of urgency for those individuals.
We asked the two interviewees about the progression of their glaucoma. One of them told us that they were in the mid-stage of glaucoma when they first visited an eye doctor, and the progression remarkably slowed after starting the treatment. They said that they can still look at the computer screen all day without issues, but acknowledged that some people cannot manage tiredness in their eyes.
The other interviewee, whose condition has progressed much further, told us that he can no longer read books or newspapers, and that watching TV is difficult without audio assistance; His eyes cannot keep tracking movements and feeling exhausted. He mentioned that he does “listen to", rather than watch NHK programs on TV. He also mentioned that due to the loss of 70% of vision in the left eye and 30% in the right eye, he uses a white cane. However, he said that steps are frightening, he can't see traffic lights, and he's had several near-miss accidents. When he goes to eat at a restaurant, he can no longer read recommended menus handwritten on the blackboard and can only rely on the standard menus at chain restaurants. He also mentioned that when ordering a new menu item, he sometimes mistakes tomatoes for pickled plums. Furthermore, it's difficult for him to distinguish people's faces, and sometimes he can’t tell someone's gender.

We asked what is necessary to change such situations.

The interviewees from GFN emphasized that glaucoma is hard to detect without an eye exam at an eye doctor. Based on that, they suggested that glaucoma detecting should be made mandatory in corporate health checkups or municipal health screenings, even though it is not currently required. They pointed out that even in comprehensive medical checkups, the results are reviewed by an internist rather than an eye doctor in some cases, meaning that aspects related to internal medicine are prioritized, and glaucoma can easily be overlooked. They stressed the importance of incorporating glaucoma detecting into health screenings and ensuring people capable of picking up on the results afterward.
Furthermore, they emphasized the need to create detecting devices that can be used easily, even by elderly individuals who may have difficulty with hand movements, especially considering that many glaucoma patients are older.

When asked about awareness activities for early detection, GFN members strongly emphasized the importance of early detection, which they only realized after the disease had progressed.
One such awareness activity is the "Light Up Green" campaign by the Glaucoma Society which aims to raise awareness before it's too late for treatment. GFN also participates in the Light Up Green campaign. They mentioned that they would appreciate proposals from young students like us. Additionally, GFN is involved in various activities, such as conducting awareness campaigns through online broadcasts. However, they pointed out that creating videos to reach a wide audience remains a challenge.

They pointed out that modern society is saturated with information, and young people have too many things to take care of, leaving little room to think about glaucoma. Even if they are told in the early stages that they show signs of glaucoma, it often gets pushed down their priority list. GFN members mentioned that many people suffer from glaucoma progression while spending time in this way. They emphasized the importance of creating criteria to raise the priority of glaucoma and reiterated the necessity of our project.

They also raised the issue of treatment continuation, which is another challenge, even after early diagnosis. They noted that after starting eye drops and lowering intraocular pressure, many people do not feel a significant change and tend to drop out of treatment midway. "A year after treatment started, about 40% of patients have already stopped treatment. Those in their primes think that it’s not changing much and let it progress... Meanwhile, the optic nerve damage that occurs cannot be recovered," they said. This highlights the importance of both early diagnosis and treatment continuation.

On a social level, they mentioned that in any country or city, the dependence on computers will continue to rise. In such a society, it is easy to imagine that the strain on our eyes will increase significantly. They believe the incidence of glaucoma will rise due to severe myopia. For this reason, they emphasized that tools like the proposed screening kits are crucial and could become widely used within 5 to 10 years.

Lastly, we were able to ask about the visual field defects experienced by GFN members. They noted that the way vision is affected varies from person to person. As a reference, they kindly provided us with images showing visual field defects. We are truly grateful for their cooperation.

Team Reflection

After interviewing GFN, we realized that our approach is both highly beneficial and meaningful. We also learned about the importance of early detection and the mental burden faced by individuals diagnosed with glaucoma. It is essential for us to raise awareness on this issue. For more details on our approach, please refer to the Education section.
Furthermore, they highlighted the potential of our project to reduce the mental burden on patients, as well as the concern that some individuals may discontinue their visits based on personal judgment. This is an important point to consider for future applications.

Dialogue with an Anonymous Government Worker

In order to get an opinion on how our project would be evaluated by the government as a whole, we decided to talk to an anonymous government worker at the Ministry of Economy, Trade and Industry (METI).

Interview

Regarding the medical costs and economic effects of glaucoma, He mentioned that He have never heard of the pressure on medical costs due to glaucoma on the government side, and He mentioned three economic effects of early detection: reduction of economic loss due to serious illness, possibility of finding other diseases through hospital visits, and the possibility of reaching out to medically underpopulated areas.
He also mentioned that it is a loss for companies in terms of productivity as well as treatment costs when young people who were able to see become blind.
In addition, he told us that it would be cheaper in terms of medical costs to find the at-risk population early and have them take medicine rather than treating them after they become severely ill.

With regard to the false positive rate, he pointed out that many people become anxious when the false positive rate is high, that this is not the way medicine should be, and that it is not a good idea to release a product at a stage when the false positive rate is high.

On the other hand, since glaucoma is not directly related to life or death, he suggested that there may be demand in areas where there are no eye clinics nearby or where people do not think of going for eye examinations. He also pointed out that Japan has a low rate of blindness and other countries have a much higher rate, so it would be a good idea to target the global market.

In addition, the applicability of this device was mentioned as detection of difficult-to-treat diseases such as cancer, lifestyle-related diseases, and brain diseases. In particular, with regard to cancer, he pointed out that cancer screening specified by the Ministry of Health, Labour and Welfare (MHLW) guidelines includes stomach, cervical, breast, lung, and colorectal cancers, most of which are examined using X-rays and require direct observation. He also talked about the fact that it is quite powerful when it comes to detecting pancreatic cancer, which is very important to detect in its early stages.

Team Reflection

Through our conversations with an official from the METI, we were able to talk about losses and medical costs for businesses. Based on these conversations, we were able to convince ourselves that our approach is one that makes sense for the country and society in general.
We were also able to hear about the potential applications of this device.

Dialogue with Dr. Shinichi Fukuda

Affiliation: Associate Professor, Faculty of Medicine and Medical Sciences, University of Tsukuba

We sought to understand the significance of a glaucoma detection device that can be used at home and gain insight into its impact from the perspective of a clinician and medical researcher through our interview with Dr. Fukuda.

Interview

Dr. Fukuda mentioned that regular eye examinations, such as those conducted during health checkups, are recommended for early glaucoma diagnosis. However, since not everyone attends regular checkups, he acknowledged the usefulness of home-based glaucoma detection. He suggested that our device could help bridge the gap between the stage when they are suspected to have glaucoma in the consequence of an eye examination and when they are in the early stages of glaucoma.

In addition to using tear fluid, Dr. Fukuda pointed out that combining a system that utilizes AI to analyze images of the eye could further reduce the risk of false positives. That, in turn, could encourage people to visit a hospital. He mentioned that the feasibility of using tear fluid depends on the sensitivity of miRNA amplification. He proposed focusing on macular degeneration, another eye disease, if glaucoma detection proves difficult.

Finally, Dr. Fukuda emphasized the importance of taking on challenges that no one has addressed before, moving forward and tackling the issue for now.

Through the interview with Dr. Fukuda, we learned that our detection device is considered valuable from a medical perspective. Not everyone attends regular checkups, but with this device, we can create a flow where individuals can detect for glaucoma at home using miRNA in tear fluid, and if they are suspected to have glaucoma, they will be ready to see the doctors. Moreover, by integrating AI-based image analysis, we may reduce the false positives rate. With further research into miRNA in tear fluid, it may even become possible to detect individuals at high risk of glaucoma in the future.

If we can achieve miRNA amplification at the femtomolar level, it would not only have a significant impact on the technology itself, but also open the door to countless applications beyond glaucoma.

Team Reflection

Through the interview with Dr. Fukuda, we learned that our detection device is considered valuable from a medical perspective. Not everyone attends regular checkups, but with this device, we can create a flow where individuals can detect for glaucoma at home using miRNA in tear fluid, and if they are suspected to have glaucoma, they will be ready to see the doctors. Moreover, by integrating AI-based image analysis, we may reduce the false positives rate. With further research into miRNA in tear fluid, it may even become possible to detect individuals at high risk of glaucoma in the future.

If we can achieve miRNA amplification at the femtomolar level, it would not only have a significant impact on the technology itself, but also open the door to countless applications beyond glaucoma.

Dialogue with Dr. Masatoshi Wakui

Affiliation: Associate Professor, Department of Laboratory Medicine, Keio University School of Medicine

Interview

First, regarding the current state of home-use POCT (Point-of-Care Testing) devices, he mentioned that the impact of COVID-19 has led to the widespread use of such devices. People purchase them at pharmacies, use them at home, take a photo of the results, and bring them to the hospital, where a doctor makes the final diagnosis. He emphasized that a doctor's diagnosis is ultimately necessary. Additionally, he pointed out that the C and T lines on these tests have become familiar to the public due to COVID-19.

Next, regarding tear fluid, he suggested that consulting an eye doctor would be better since tear fluid is not commonly used for testing. While tear fluid is minimally invasive, there is a risk of damaging eyes if not collected improperly, so we should consider how the general public can safely collect it. He also mentioned that the composition of tear fluid is not complex and that its low oxygen content helps maintain a stable pH, which is an advantage compared to the unstable composition and pH levels of urine.

Regarding diseases with disease-specific nucleic acid biomarkers, he mentioned that cancer is intensively investigated. Then, he shared an example of a system where patients collect urine and send it to a company for analysis. Since this is not covered by insurance and is not certified as a medical test, he said another medical treatment is required. The reason why it is not certified as a medical test is that the system does not causesubjective symptoms like pain or itching. However, for cancer-related POCT, even if the kit returns a positive result, follow-up tests like CT scans and endoscopies is still difficult due to its cost. He told us that discussions are ongoing with doctors about how to bridge the gap between such tests and insured medical care. In our case, he suggested that we should also consider Ease of moving on to the next step after using our device.

Regarding the data required by the Ministry of Health, Labor, and Welfare and third-party certification bodies, he mentioned that the sample size required depends on the pilot study. In general, for common diseases, the required number of participants for experiment increases. It was also noted that the time required for approval varies depending on the urgency of the disease, and that approval times may fluctuate based on the government's budget situation. He suggested that eye doctors are more familiar with the Ministry's requirements regarding eye conditions, so we should consult them.

Finally, in response to the question of what users expect for POCT devices, he raised the following points:

• First, it is important to provide clear instructions on the collection of tears so that users can easily understand the process.
• Next, the size of the device should be about the same as a COVID-19 test kit, and it should not be too small. Additionally, since users will bring the device to an eye doctor, it should be portable.
• Moreover, he mentioned that maintaining a constant temperature of 37 ℃ at home should be easy.
• Finally, he pointed out that we must explain whether the device would still work properly with poor-quality tear fluid, such as the "watery" tears associated with hay fever (allergic conjunctivitis).

Team Reflection

Through the interview with Dr. Wakui, we understand better the current state of POCT devices. He suggested that the Lateral Flow Assay, familiar with COVID-19 test kits, be potentially beneficial. That provided us with insights that will lead to further interviews. We also discussed the necessary conditions of biological samples and the potential risks of damaging the eye, which will require careful consideration.
Moreover, we reconfirmed the importance of including the information that users truly need. As for the size, we decided to aim for a device weighing around 10 g and measuring approximately 70 mm x 30 mm, the same size as COVID-19 detection kits.

Dialogue with Professor Toru Nakazawa

Affiliation: Professor, Ophthalmology, School of Medicine, Tohoku University

After discovering a paper on the profiling of miRNA in tear fluid, we attempted to contact the first author to inquire further about the research. However, the first author had recently relocated and their contact information was unavailable. As a result, we decided to reach out to the corresponding author listed on the paper, Prof. Nakazawa.
Prof. Nakazawa is a leading authority in ophthalmology at Tohoku University. Through an interview with him, we aimed to identify the specific miRNAs that should be detected.

Interview

Prof. Nakazawa pointed out that the optic nerve, which is affected by glaucoma, and tear fluid are anatomically separated. Therefore, significant changes in miRNA concentration in tear fluid might not occur in glaucoma cases. He also mentioned that miRNA circumstances can vary significantly due to age, physical constitution, lifestyle habits, etc. Since glaucoma is a progressive disease, identifying biomarkers that change with the disease's progression is essential, but such biomarkers have not yet been discovered.
Additionally, he suggested that since miRNA can also be affected by oxidative stress, it might be more effective to directly measure oxidative stress itself.

Regarding the variations in miRNA concentration across different studies, he explained that these differences are often due to measurement discrepancies between laboratories. However, he felt confident that using the miRNA identified in the same research should not be problematic.

When asked why tear fluid samples are rarely used, Prof. Nakazawa explained that aqueous humor and blood are typically collected instead, as obtaining tear fluid is less common. Obtaining aqueous humor involves using a needle, which often requires patient consent and limits the number of samples collected. He further noted that while tear fluid is concentrated and suitable for disease testing use, individuals with dry eyes have less tear fluid, which could result in an artificially elevated initial concentration.

Based on this discussion, he emphasized the need to establish baseline miRNAs or proteins that do not change between glaucoma patients and control groups. Having a standard in place would be crucial for effective screening. He also suggested that during screening, it would be essential to collect aqueous humor, blood, and urine samples as well. Furthermore, he proposed using proteins like lactoferrin in tear fluid as potential biomarkers.

Team Reflection

We reconfirmed the significant challenges in using miRNA in tear fluid as biomarkers due to the lack of definitive data, the variability in miRNA levels based on age, constitution, and lifestyle, and the progressive nature of glaucoma, for which no associated miRNA changes have yet been identified. However, overcoming these obstacles could have a substantial impact.
Moreover, once suitable miRNAs are identified, it will be critical to establish a standard biomarker to account for potential variations, making this an important next step in our research.

Dialogue with Dr. Takenori Inomata

Affiliation: Associate Professor, Department of Ophthalmology, Juntendo University School of Medicine

Dr. Inomata is an expert in tear fluid.
We conducted an interview with Dr. Inomata to confirm whether miRNA in tear fluid can really be recovered with Schirmer strips and whether miRNA can be properly recovered from tear fluid.

Interview

He said that using Schirmer strips is a good point of view, although it remains to be seen whether miRNA can be obtained from them.
It was also pointed out that when general users collect tear fluid, it is important for us to ensure that the act does not fall under medical procedures.He also pointed out that it is difficult for general users to collect tear fluid themselves when using Schirmer test at home. It is appropriate to use capillary action after eye drops when collecting at hospitals, etc., but it may be very difficult to perform this technique at home, or it may fall under the category of medical practice and be prohibited.

In addition, when asked whether miRNA is contained in tear fluid, he said that he would not be able to tell without trying.
Furthermore, he told us that he did not know the type or concentration of miRNA in tear fluid exosomes, and that he could not say anything without trying.

We also asked about the method of destroying tear fluid exosomes and whether it is good to use surfactants, but were told that we would not know until we try, but that they use exosomes when looking for biomarkers of breast cancer in tear fluid.
He also said that he didn't know about the variation in miRNA levels from type to type and from individual to individual because he was not a specialist.

As for glaucoma-specific miRNAs, we were told that he did not know because researchers are in the process of searching for them right now.
He said that the validity of a biomarker can only be validated through a thorough clinical trial. Finally, he said that he would provide us with tear fluid samples if we wanted them for our tear fluid screening.

Team Reflection

We reconfirmed that we can't know what we will get unless we try experiments. Also, the validity of a biomarker can be said to be valid only when it is verified through a solid clinical trial, and a small-scale survey is not reliable. We need to conduct large-scale studies to identify miRNAs.

Dialogue with Prof. Takahiro Ochiya

Affiliation: Professor, Division of Molecular and Cellular Therapy, Medical Research Institute, Tokyo Medical University

We approached Prof. Ochiya, a leading researcher in miRNA-based cancer diagnostics and a pioneer in creating ventures for the societal implementation of his research on cancers like pancreatic cancer, to inquire about the challenges of detecting miRNAs as biomarkers for glaucoma.

Interview

Prof. Ochiya explained that he has successfully commercialized ventures related to pancreatic cancer diagnostics and is involved in joint research on various exosomes. He emphasized that extracting exosomes from any bodily fluid and analyzing the contained proteins would offer higher sensitivity and specificity. While collecting exosomes from tear fluid is one possible approach, he suggested that it might not be necessary to extract them, as their contents often leak out after the exosomes break apart. He also mentioned that using Schirmer strips to collect exosomes could be a good method.

Regarding miRNAs, Prof. Ochiya noted that while they can serve as biomarkers to some extent, many studies lack reproducibility, making their reliability questionable. This variation, he explained, is influenced by factors such as how the bodily fluids are collected, storage time, and the conditions of the samples. He stressed the importance of stabilizing these factors to achieve consistent analysis results.

He also mentioned that although many researchers are trying to use AI for meta-analysis across multiple datasets to determine miRNAs bioinformatically, this technique has not yet been fully established. Despite numerous efforts, this method has not been successfully implemented.

On using blood as a biological sample, Prof. Ochiya highlighted that miRNA concentrations in the blood tend to be diluted due to numerous substances, making tear fluid a superior option. However, he pointed out that disease-derived miRNAs are extremely minute, making their detection particularly challenging.

Prof. Ochiya suggested conducting a pilot study with an eye doctor to narrow down the candidates through a few experiments, and then large-scale screening to identify specific miRNAs. He also recommended considering age differences when collecting samples, as age is likely to affect the results, and that it would be beneficial to standardize the age within the group. Even with these considerations, he mentioned that completing this research, even with expert involvement, might take about three years.
He mentioned that while it would be ideal for the detection device to have a visual display like COVID-19 testing devices, this might be difficult to achieve.

In conclusion, Prof. Ochiya emphasized, "The most challenging part is selecting the miRNAs. This is where misinformation can occur, and you must avoid that." He also kindly offered to verify the reliability of any papers we send him.

Team Reflection

While our project has repeatedly been recognized as challenging, both by our team and during external evaluations, our discussions have led us to realize the importance of simplifying the system wherever possible. Until now, we had primarily focused on free miRNAs in tear fluid. However, learning from an expert that miRNAs within tear exosomes are more important allowed us to adjust our approach.

According to a liquid biopsy expert, using tear fluid as a biological sample is highly favorable, and this presents a substantial advantage for our project. On the other hand, there are concerns regarding the reliability of using miRNAs as biomarkers, and we must carefully consider the experimental conditions and the standards for comparison. Although this is highly challenging for non-experts like us, we are fortunate to have received cooperation in this area. Prof. Ochiya also suggested conducting a pilot study with eye doctors.

Above all, we must remain honest and dedicated to science. Our conversation with Prof. Ochiya reaffirmed the importance of acting with integrity and avoiding any falsehoods in our research.

Dialogue with Dr. Ken Komiya

Affiliation: Japan Agency for Marine-Earth Science and Technology, Researcher, Supar-cutting-edge Grand and Advanced Research

Dr. Komiya developed a new isothermal amplification reaction, the L-TEAM reaction, when he was previously at the Tokyo Institute of Technology, enabling isothermal amplification at a low temperature of 37 ℃.
We thought about using this technology to detect fM order miRNA and headed into the interview.

Interview

The interviews were conducted over four sessions.

First Interview

To determine the amplification system, we went to talk to Dr. Komiya, who was working on the ‘L-TEAM’, a type of SDA. Our goal is to amplify and quantify the extremely small amounts of fM order miRNA. If we don't carefully construct the reaction system from the early stages of project design, we have the risk of repeating unnecessary experiments. Therefore, we planned to consult with Dr. Komiya, who is well-versed in the SDA, to gain insights into its practical application. We also aimed to receive feedback on the reaction system we were considering and the reasons for our selections.

Contents:
First, we learned about the differences between PCR and SDA. PCR, a common method for nucleic acid amplification, achieves high accuracy by controlling temperature cycles at high temperatures over time. In contrast, SDA operates at relatively low temperatures and does not control temperature cycles, resulting in lower specificity. Because of this, PCR is generally preferred, and Dr. Komiya emphasized the importance of clearly defining our purpose for using SDA.

Dr. Komiya mentioned that targeting miRNA in tear fluid has advantages over blood. Tear fluid contain fewer proteins and other substances than blood, which can coagulate above 42 ℃ and hinder miRNA detection. Therefore, using tear fluid may allow for a shorter and simpler process, as it eliminates the need for extensive sample preprocessing.

As mentioned above, SDA amplifies nucleic acids through chemical reactions, which emphasizes that amplification occurs statistically. Additionally, Dr. Komiya pointed out that the performance can vary significantly based on the quality of the DNA polymerase and nickase used. Since the expression levels of biomarker miRNAs are not zero in the control group, establishing a system to detect their presence is insufficient. Furthermore, as the concentrations are on the order of fM, we are attempting something quite challenging. It is also necessary to consider whether to amplify the signal linearly or exponentially when designing the system.

Although it is a very challenging project, Dr. Komiya emphasized that if successful, it could have a significant global impact, encouraging us to persevere.

Team Reflection 1

We plan to use SDA instead of PCR, keeping in mind the potential for home use. However, it is extremely challenging to establish a system that can specifically amplify miRNA on the order of fM. I realize how essential it is to set clear goals as we move forward with the project within our limited timeframe.
By thinking backwards from our goals when setting goals, we can narrow down the conditions required for the amplification system. We decided to develop a more specific amplification system and later discuss it again with Dr. Komiya.

Second Interview

Based on evaluations from other stakeholders, we recognized that the top priority condition is the ability to amplify miRNA from tear fluid "at home".
We developed a concrete miRNA amplification system that can be used at home and created the corresponding experimental protocol. We returned to Dr. Komiya to receive feedback on our work and to learn about the characteristics of the reagents we would use, as well as the experimental techniques. Additionally, we inquired about the features of Dr. Komiya's research on "L-TEAM" to see if we could incorporate any of its aspects into our amplification system.

Dr. Komiya mentioned that there are variants of SDA, each with its strengths and weaknesses. While it is not possible to definitively determine which method is optimal, he noted that using CRISPR-Cas after SDA amplification could be effective for detecting the very low concentrations on the order of fM.
Dr. Komiya emphasized that enzymes play a crucial role in achieving sensitivity with SDA. He mentioned that non-specific amplification should occur due to the interaction between DNA polymerase and nickase. Additionally, he noted that performance can vary between different enzyme lots, making it challenging to replicate sensitivities comparable to previously reported results. In this regard, SDA is less robust than PCR.

Dr. Komiya mentioned that in a recently published system[^1], they successfully delayed the onset of non-specific amplification by stacking simple SDA in two or three stages. While this approach is slower than the exponential amplification provided by EXPAR, it may reduce concerns about leaks and contribute to avoiding false-positives in detection devices.

Dr. Komiya emphasized the importance of being especially cautious about contamination during experiments. He explained that aerosols from amplification products or residues on lab equipment can lead to unexpected results. In exponential amplification, contamination can easily compromise the validity of the experimental outcomes, necessitating the utmost care throughout the experimental process.

Team Reflection 2

We envisioned a system for developing a device that could be used at home, aiming for isothermal amplification methods that do not require temperature fluctuations or specialized techniques. However, we realized that we do not know which amplification method is optimal until we start experimenting. Avoiding non-specific amplification is challenging, so selecting a system with minimal leaks is crucial. From then on, we would conduct experiments with careful attention to contamination and adopt amplification reactions that can be utilized for our desired amplification system. Our goal is to subsequently connect this to CRISPR-Cas for further amplification.

Third Interview

Continued from last time, we decided to design a detailed protocol for the amplification system and seek further advice. Since this type of experiment is not conducted in our lab, we aimed to delve deeper into how to interpret and analyze the data we would obtain.
Additionally, we noted that the biomarker miRNA is expressed in both the control and patient groups, and ultimately, we need to incorporate quantification into the amplification system. We also asked about the feasibility of achieving this.

Dr. Komiya explained that for quantifying amplification products, intercalators are used, and since we will quantify the products using Real-Time qPCR, direct comparisons between different wells should be avoided. He advised setting a threshold at the onset of the fluorescence curve and performing a global quantification to circumvent this problem.

Dr. Komiya mentioned that increasing the number of steps in the amplification system does not necessarily lead to greater signal intensity. In fact, having more steps can hinder the progress of reactions due to interference between steps and the overall congestion within the system. Therefore, he emphasized the importance of establishing a simpler amplification system to effectively advance the project within our limited timeframe.

Dr. Komiya explained that non-specific amplification can impact quantification. He noted that due to the two enzymes being used, errors can occur even at target concentrations around 1 pM, and overcoming this challenge is essential for detecting fM levels. He suggested that at the beginning of the amplification system, we should slow the reaction rate to suppress the onset of the negative control, allowing for a time gap in the reaction. Finally, he recommended using CRISPR-Cas for detection to improve the results.

Dr. Komiya also provided advice regarding the use of tear fluid samples. He explained that actual tear fluid contains various contaminants in addition to miRNA, meaning they are not a pure environment. Therefore, he suggested a phased approach for evaluating the amplification system: starting with a synthetic environment containing only the synthesized miRNA, then introducing the synthesized miRNA into artificial tear fluid, and finally using actual tear fluid. Additionally, he mentioned that miRNA is often found in exosomes within tear fluid, which can be disrupted using surfactants. He also noted that ethical approval would be necessary for using real tear fluid samples in our experiments.

Team Reflection 3

Quantification requires a well-established experimental environment, and we must avoid contamination at all costs. While non-specific amplification may be unavoidable, it might be possible to mitigate its impact through careful design of the amplification system, similar to the L-TEAM approach. On the other hand, connecting too many reactions could negatively affect sensitivity, so we aim to develop a simpler system.

We also focused on creating a clear and concise protocol to ensure reproducibility, aiming to write it in a way that anyone can replicate the experiment. Since using actual tear fluid necessitates ethical approval, this step is essential for validating our system. Therefore, we plan to submit an ethical application regarding the use of tear fluid to the ethics review board at our research institution.

Fourth Interview

We decided to conduct experiments on the amplification system and share the results to receive feedback on current problems and future directions. Our goal was for Wet Lab to address questions regarding experimental results and non-specific amplification from Wet Lab, while Dry Lab obtaining feedback on the proposed SDA model. Additionally, we inquired about the sequence design for using a three-way junction complex in SDA to enhance specificity.

We received advice regarding various reactions, including HCR and EXPAR. It was pointed out that we might be making inferences about things not directly observable from the experimental results, which could lead to misunderstandings. Dr. Komiya emphasized the importance of being cautious about this. Additionally, he suggested that clearly stating the lengths of the sequences used in the experiments on our presentation slides would make it easier for the audience to understand.

We discussed how the annealing steps affects the specificity in HCR, based on the results we obtained. While conducting various experiments with EXPAR, we encountered results that contradicted previous studies. In such cases, we were advised to critically examine and reflect on our own findings. Initially, we had considered a system combining EXPAR and HCR, but Dr. Komiya suggested rethinking this approach, as it could become overly complex and undermine the advantage of being enzyme-free.

We also received answers to several questions regarding our experiments. Dr. Komiya explained that the correlation between the amplification curve and the target concentration holds only up until the template is exhausted and the curve reaches the plateau, and that discussions should focus on the inflection point. While it is possible that the plateau value could shift by 20-30%, if the deviation exceeds 50%, it would be wise to suspect an error. He also mentioned that nonspecific amplification is likely caused by ab initio synthesis, which is unavoidable, and advised that we should design the system to minimize its impact as much as possible.

We also received advice regarding Dry experiments. While we were considering optimizing the enzyme using a model, Dr. Komiya pointed out the challenge due to the large variability between different lots. He also advised us to keep in mind that, no matter how precise the model is, it is difficult to determine whether it accurately reflects what is happening in the real system.

Team Reflection 4

It has been decided that we will likely not use HCR. Moving forward, we plan to proceed with experiments focusing on TWJ-EXPAR.
We have arranged the consultation with Dr. Komiya again when selecting the amplification method. However, it would be best to adopt a system that we have tested and found to actually work successfully.

In Dry Lab, our initial goal was to model EXPAR in order to optimize the polymerase and nickase. However, based on the Wet Lab results and the recent advice, we decided to narrow our focus to optimizing the template concentration. We will now shift towards fitting simulation results with experimental data, as well as developing higher-order EXPAR models, designing sequences, and evaluating specificity.

Dialogue with Prof. Masayasu Kuwahara

Affiliation: Professor, Department of Chemistry, Nihon University College of Humanities and Sciences

In our quest to determine the optimal amplification system for home detection of miRNA in tear fluid, we went to see Dr. Kuwahbara, who, like Dr. Komiya, is an expert in the isothermal amplification method and the developer of the SATIC method.

Interview

He told us that there are multiple methods of amplification of nucleic acids, and he taught us about several orthodox amplification methods.

First, he advised us about the system to quantify the signal by Lateral Flow after cleavage of ssDNA by SDA and CRISPR-Cas. He gave us a brief lecture about SDA and helped us understand more about it.
He gave us a brief lecture on SDA, which helped us understand more about it. The part of SDA that recognizes target is also key in SDA, the professor said. He said that TWJ-SDA and SATIC, which form a three-way junction complex to initiate the reaction, have very high specificity. In addition to amplification rate, specificity is also necessary for the amplification of fM-order miRNAs in tear fluid, and he suggested that a robust system could be designed by focusing on this aspect as well.

On the other hand, he said that it is still difficult to detect fM-order miRNAs, and this is an issue that not only iGEM but also real researchers are working on. He emphasized that it is essential to build a robust system.

As for how to proceed with the experiment, he advised us not to go for detection of extremely low concentrations from the beginning, but to conduct the experiment at a relatively high target concentration, and based on that, improve the system and gradually lower the concentration. He also said that we need to consider the performance of each polymerase product, so we should use one that has a proven track record in use and remember to keep the lot number in mind.

He told us that it is important to note that it is only an auxiliary tool since it is the doctor who ultimately diagnoses glaucoma.

Finally, he introduced Dr. Yoshimi, who is affiliated with the Institute of Medical Science.

Team Reflection

We thought We had devised something more specific about the amplification method than last time, but it turns out that We need to make it more detailed.
Quantification of miRNAs, which only differ by a few fM and 10-fold, is still difficult, and the key to this project is how to overcome this difficulty. Therefore, we need to do more research on the amplification method and also do more interviews with experts to get their feedback on the system we are devising.

Dialogue with Prof. Tomoji Mashimo

Affiliation: Professor, Division of Advanced Animal Genomics, Institute for Experimental Animal Research, The Institute of Medical Science, The University of Tokyo

To create a robust amplification system to amplify deficient concentrations of miRNAs in tear fluid, we explored the potential of CRISPR-Cas. Therefore, we decided to talk to Prof. Mashimo, who developed Cas3, the first CRISPR-Cas in Japan.

Interview

Two interviews were conducted. In addition, an interview was conducted with Dr. Yoshimi, who belongs to the same laboratory as Prof. Mashimo and was introduced by Dr. Kuwahara.

First Interview with Prof. Mashimo

Based on the knowledge gained from the development of CONAN, a Cas3-based COVID-19 detection system, we attempted to discuss various issues, from the difficulty of low-concentration detection to the final product design.

First, he pointed out our devised system was too complex. He said that although it was necessary to increase the amplification rate, it was difficult to achieve this by simply combining amplification systems, and a major gap with intuition lurked. For example, he said that methods using microfluidic devices such as microreactors are excellent as simple detection systems and even single molecule observation is possible, but had not yet been used at home.

MiRNAs are as short as 20 nt and are difficult to amplify by simple PCR. Among SDAs, LAMP used in CONAN, for example, has the advantage of high sensitivity and specificity because it uses 4-6 primers and the reaction proceeds at 65 ºC, he said. He pointed out that SDA without LAMP would require some innovations to increase specificity.
CRISPR has recognition sequences, and the activity varies depending on the sequence. Therefore, he said, we had better use the sequence with the highest activity. In terms of the form of the signal, he said that LFA (Lateral Flow Assay), which uses strips like CONAN, is simpler than fluorescence observation from the perspective of home use. However, he also said that the sensitivity would be reduced in terms of not using expensive detectors, so it would be necessary to amplify the signal intensity with specificity using an amplification system.

The project itself is promising and interesting, but, at the moment, there is a problem with amplification sensitivity, and a simpler and more robust system needs to be designed, he said.

Mashimo Lab promised to provide us CRISPR-Cas3.

Team Reflection 1

We needed to analyze the strengths of existing methods such as LAMP and assemble an amplification system with high sensitivity and specificity. However, the amplification system we were then designing is overly complex and it would be wise to find a simple system. For this purpose, it is necessary to continue discussions both within the team and at Human Practice. We then decided to start the experiment after the completion of the system. CRISPR signal enhancement and conversion will be needed to design a home inspection device. We decided to talk to Dr. Yoshimi of the Mashimo Lab about this next.

Second Interview with Prof. Mashimo

We revisited Prof. Mashimo to ask for advice on the amplification system and for his knowledge of CRISPR-Cas, which would be utilized in the amplification and quantification part. We also sought to learn about the skills and points to keep in mind when conducting the experiments.

Before the content, he advised us on the policy of the detection device we were aiming for. He said that since only doctors are allowed to perform diagnosis, what we can do is only detection and that the user needs to visit the doctor for collection. It was desired that the device would encourage users to visit the hospital.

We also organized the usability priorities. He said that the device should be as simple as possible for home use, not require special machinery for temperature change, and importantly, the results should be easy to read.

There are several CRISPR-Cas with collateral activity, including 3, 12a, and 13, and he said that 3 and 12a are suitable for our amplification system. Both 3 and 12a have similar performance, but 3 has higher specificity and lower speed than 12a. He advised us that Cas3 might be better for our system, where sensitivity is important.

Apart from that, it will be important for the device itself to be environmentally friendly in the future. An environmentally conscious design is not only environmentally friendly, but may also give the public a good impression of the product and make them more likely to purchase it. Therefore, we were advised to put thought into the device's appearance as well.

Team Reflection 2

We were able to hear a little about Cas but were unable to meet Dr. Yoshimi this time.
We realized that we had become so absorbed in the discussion of amplification systems that we had drifted away from the philosophy of prioritizing the users. Here, we decided that we should return to the users’ point of view and design a device that is simple and easy to use. In addition, since we planned social implementation, we should pay attention not only to the interior but also to the exterior appearance, and by launching a product that is in line with social trends, we may achieve commercial success.

Interview with Dr. Yoshimi

To hear about CRISPR-Cas, we also spoke with Dr. Yoshimi, an associate professor in Mashimo's lab.

He said that it is possible to run CRISPR-Cas based on dsDNA amplified by SDA, similar to CONAN, but that the amplification sensitivity and specificity must be enhanced to some extent on the SDA part. He also pointed out that Cas3 has the advantage of higher specificity than 12a and may be able to remove the noise in the SDA part where NC rises in our system. He pointed out that to reduce the false positive rate, we need to eliminate the rise of NC as much as possible. He advised us to try to do experiments with both proteins, as there is no way to know without actually doing the experiments.

He said that it is possible to activate Cas3 with dsDNA amplified by SDA because Cas3 can be fully activated with about 60 bp of dsDNA containing the recognition sequence. On the other hand, he said that he was not sure if it would work with one-pot. The one-pot that was initially assumed may cause the template used for SDA to be cleaved by Cas3 activation, and this should be discussed as well. In addition, he suggested that it is better to experiment with both two-pot and one-pot because it is not known whether they work properly due to the degree of crowding and the presence of other polymerases and nickases.

We were discussing the quantification using SYBR Green, but he suggested that we adopt FQ probes when using Cas because SYBR Green is not reliable enough, and also because of the congestion in the system. Also, since Cas3 runs at 37 ºC, they need to pay more attention to temperature control during the experiment than before.

Team Reflection 3

We had decided to use an amplification system that uses SDA followed by CRISPR-Cas, but we decided to let the results of the experiment determine whether to use 3 or 12a. We are considering using the one that reacts in a shorter time and has better specificity. Also, which is better, one-pot or two-pot, is unknown until the experiment is done, so we decided to set up two experimental systems and compare them. From here, we need to discuss based on the results of the experiments. We decided to visit him as an outside advisor again after the results are available. At this time, we were provided with reagents to use in the CRISPR-Cas3 and 12a experiments.

Dialogue with Dr. Makoto Aihara

Affiliation: Professor and Chair, Department of Ophthalmology, Faculty of Medicine, University of Tokyo

Having completed the foundation of amplification methods to some extent through interviews, experiments, and mathematical models, our next task was to discuss miRNAs that have a causal relationship with glaucoma. To do so, we decided to speak with Dr. Aihara, a leading authority on glaucoma.

Interview

Since the amplification part seems to be progressing well, he mentioned that from now on, the priority would be to identify miRNAs that are truly causally related to glaucoma. Regarding miRNAs associated with glaucoma, he noted that biomarkers derived from the optic nerve head currently being highlighted are likely to emerge after the progression of glaucoma, and that further research is needed for early-stage biomarkers. He also pointed out that since it is possible to observe early-stage glaucoma with a fundus examination, a marker that is equally or even earlier detectable is needed. Additionally, it was suggested that we investigate markers that cause an increase in intraocular pressure.
As for concerns regarding tear fluid collection, it was indicated that debris can get into the tear fluid during collection, so care must be taken. It was also pointed out that collecting tear fluid by oneself is surprisingly difficult.

Furthermore, one of the strengths of fundus examination is that it allows for the identification of individuals who are structurally predisposed to glaucoma just by looking at the images. However, it was critically noted that if patients don’t undergo fundus examination, it may be meaningless. Moreover, those who use a glaucoma detection device at home would likely visit an eye doctor in the first place. On the other hand, he also mentioned that being able to use such a device at home is quite appealing, as it could encourage people to visit an eye doctor.

As for potential applications of the detection device, it was suggested that a multi-type device, which can detect not only glaucoma but also other diseases such as lifestyle diseases, would be better suited. It was also mentioned that a device might be more suitable for detecting single-factor diseases than multi-factor diseases like glaucoma.

Finally, he indicated that the demand for such a device might be higher in countries with lower medical standards than in those with higher standards. Additionally, it was pointed out that the most problematic form of glaucoma in Asia is angle-closure glaucoma, which can lead to a rapid increase in intraocular pressure and the progression of symptoms.

Team Reflection

Since it is possible to observe early-stage glaucoma with a fundus examination, it is necessary to find markers that are equally or even earlier detectable. When investigating biomarkers ourselves, we must create an action plan that considers the advice received, including looking into markers that cause an increase in intraocular pressure.
As many researchers have mentioned this, it has also been pointed out that collecting tear fluid by oneself is surprisingly difficult. This aspect will require further consideration. Furthermore, we were able to reaffirm the strength of our approach - providing a device that can be used at home - which can serve as an opportunity for individuals to visit an eye doctor. Having reconfirmed our strengths, we plan to incorporate the feedback regarding its potential applications as we move forward with future interviews.

Dialogue with DeepEyeVision

DeepEyeVision, Inc. is a company that uses AI technology to provide diagnostic assistance for ophthalmic diseases based on fundus images.
Based on Dr. Fukuda's opinion that combining fundus examination with AI could improve accuracy, we tried to learn what kind of contribution we could make toward glaucoma detection by combining with dry examination through dialogue with DeepEyeVision.

Interview

First, he mentioned that dry technology had progressed enough to predict various ophthalmologic diseases, such as corneal shape abnormality, including glaucoma, from fundus images.
He also pointed out the difference between diagnosis and examination in Japan. In Japan, glaucoma is diagnosed only by eye doctors, and the certification process for POIROT and AI is long and difficult, so it is necessary to use the term "examination" rather than "diagnosis".

He also said that the suspicion of glaucoma was currently handed down in medical checkup, in addition to seeing an eye doctor. However, he said that it is rare for people in the prime of their working lives to go to the eye doctor regularly or go checkup because of the lack of subjective symptoms in the early stages of glaucoma. It is also difficult to get fundus photographs at eyeglass shops due to multiple regulations, which makes it difficult to suspect glaucoma outside of ophthalmology.

Therefore, he said that if a technology that enables early detection of glaucoma by wet examination exists, it will contribute to people's health and there is a great need for such a technology. In Japan, the number of patients is increasing due to the aging of the population and the possibility that rural areas will be abandoned because eye doctors are concentrated in urban areas due to urbanization cannot be denied. So, early examination technology from a wet device will be important for these people. He also pointed out the possibility that for some elderly people, an analog method may be easier to believe in than a digital one.

As we discussed this together, a commercial strategy emerged: people who are suspected of having glaucoma through AI testing could use POIROT on a regular basis instead of going to the hospital. Until now, we had thought that POIROT would be an incentive to visit an eye doctor, but we believe that not only that, but that ongoing testing could reduce the burden of hospital visits.

Team Reflection

Early diagnosis of glaucoma leads to maintenance of quality of life. For this purpose, there are methods to approach the disease not only from the wet side but also from the dry side. The more testing options available, the better.
Therefore, we have found that the use of POIROT together with dry technology can reduce the false positive rate and hospital visits. In addition, we are now at the stage where we need to consider how to market the product for commercial development, which requires reliability of the product itself, apart from the false positive rate.

Dialogue with Dr. Emi Inagaki

Affiliation: Research Fellow of the Japan Society for the Promotion of Science (Project Assistant Professor), Department of Ophthalmology, Department of Physiology, Keio University School of Medicine

We were in the process of submitting an ethics application for our project when we realized that there was no eye doctor among the applicants. Therefore, Dr. Inagaki, an eye doctor who had been working as a special research fellow in our lab, agreed to handle the matter.
At that time, we saw this as an opportunity to have our project evaluated from the perspective of an eye doctor, so we decided to ask about matters beyond the ethics application as well.

Interview

Dr. Inagaki mentioned that using biomarkers in tear fluid is a very positive approach from the user's perspective. She also emphasized that since glaucoma cannot be easily tested at home, our project holds significant value.

However, she advised that since a definitive biomarker for glaucoma has not yet been discovered, it might be worthwhile to investigate other diseases or biological phenomena such as aging. She pointed out that the flexibility of this system is that it could be applied to other targets, not just glaucoma. For example, there may be demands for targeting individuals who are unaware of their condition and don't visit hospitals but exhibit clinical findings, such as those with VDT syndrome. However, she also stressed the need for establishing causal relationships and evidence for miRNAs.

In relation to the ethics application, she explained the importance of using PCs during experiments with tear fluid. For dry eye, lactoferrin is used as the PC, and she mentioned that we would need to identify a suitable PC for glaucoma as well. She suggested the possibility of using animal models, such as mice, instead of relying solely on human glaucoma biomarkers. Additionally, she pointed out that, as expected, we must avoid false positives when designing the detection device.

Initially, we planned to prepare around 10 samples of actual tear fluid, but Dr. Inagaki advised that it would be better to have approximately 50 samples to validate the system properly.

Team Reflection

Based on Dr. Inagaki's advice, we decided not to limit the application to glaucoma but to broaden the scope to cover ophthalmic diseases in general. This reaffirmed that the versatility of our project, beyond just glaucoma, is one of its strengths.
Additionally, when conducting experiments using actual tear fluid, it will be essential to set an appropriate positive control (PC). We have decided to carefully consider the selection of this PC as well.

Dialogue with Dr. Shimizu of OUI inc.

To develop a commercial strategy for the commercialization of POIROT, we decided to talk to Dr. Shimizu, an eye doctor and president of OUI inc, a company that operates a remote diagnosis business.

Interview

Regarding our system, he said that not only would it be difficult to explain during product development, but also because the public is primarily interested in the fact that a glaucoma detection device has been created, there is no meaningful reason to claim it as synthetic biology. He also praised us for focusing on early detection and early treatment. Apparently, many people have benefited from early detection. Furthermore, it was pointed out that such a device could be beneficial in Japan, where people are less likely to visit eye doctors compared to other countries. Regarding the collection of tear fluid using Schirmer strips, he pointed out that this would be quite difficult. With usability in mind, he suggested something like using an eye wash solution to wipe the eye and then collecting that liquid. He mentioned that POIROT's amplification system is relatively simple, and if it's not continuously improved as a product, competitors might emerge. Therefore, improving POIROT's functionality would be one of the most important strategies.

Specifically, he suggested the following:

• Improving operability: We had assumed POIROT would use Schirmer strips for tear fluid collection even after commercialization, but this isn't suitable for easy use by everyone. For example, he suggested possibly collecting miRNA from tear fluid using something like an eye wash solution.
• Expanding the target: Currently, POIROT is optimized for detecting glaucoma, but due to its flexibility, it could potentially detect various diseases with disease-specific nucleic acid biomarkers. By predicting the risk of multiple diseases with one device, we could increase the number of potential users.
• Developing separate services: For example, we could improve usability by developing a service that helps users who show a risk of glaucoma using POIROT to book appointments at nearby eye clinics.

He mentioned that to gain market share, proper evaluation and reliability are necessary. While healthcare systems differ between countries, he pointed out that we could increase profits by determining the product price based on market size.

He explained that product reliability leads to purchases, and suggested we need to start by lobbying at academic conferences to get experts to recognize POIROT as an evidence-based device. Simultaneously, he said patenting POIROT's system is meaningful, and that patents are important not only for fundraising but also for system reliability.

He also mentioned the need to strategize about the product's sales format. We need to decide where it can be purchased and used - hospitals, clinics, pharmacies - and whether it should be classified as a pharmaceutical product.

In the future, he suggested consulting with the Medical Devices Division of the Bureau of Social Welfare and Public Health, Tokyo Metropolitan Government to determine the appropriate classification. For sales, he suggested that besides direct consumer purchases, introducing it to corporate health check-ups could generate stable annual revenue.

Regarding responsibility for dual-use or misuse, he said appropriate disclaimers would suffice. However, he warned we should be careful about false positives originating from our product itself.

As an eye doctor, he expressed that what he hopes for from POIROT is to identify more patients.

Team Reflection

This provided a good opportunity to consider POIROT's future potential. To maintain a continuous business, the product must keep evolving. This involves not only improving performance, such as reducing false positive rates and expanding detectable targets, but also enhancing usability aspects like tear fluid collection methods. Moreover, trust from experts is crucial when dealing with medical devices. By lobbying through academic conferences and gaining third-party recognition as a reliable product, we can make it more user-friendly. Rather than vaguely considering sales, we can maximize profits by setting prices that match the market and carefully selecting sales methods and locations.
We also confirmed the necessity of obtaining patents and consulted with the intellectual property management team at the University of Tokyo.

Dialogue with Dr. Shun Sakuraba

Affiliation: Senior Researcher, Quantum Life Science Area, National Institute of Quantum Science and Technology

Since the binding reaction between target and template was found to be a rate-limiting reaction, we thought it was important to analyze the binding rate, i.e., to determine the rate constant of the binding reaction. Therefore, we decided to examine how much the reaction rate constant changes when a single nucleotide mismatch is inserted in EXPAR and TWJ, respectively, from the dry side. We thought that Molecular Dynamics Simulation (MD Simulation) could be used for the verification on the dry side. For this purpose we went to talk to Dr. Sakuraba, an expert in the computational science of nucleic acids.

Interview

When we asked if we could use MD Simulation for our analysis, he told us that it would be difficult to find what we wanted to study with MD Simulation. It is technically difficult to do MD Simulation with only a few months left, and we found that what we wanted to investigate this time could not be handled by a messy MD Simulation. After discussion with the professor, we decided to investigate the sequence specificity of EXPAR and TWJ using the difference of free energies. We also discussed alternatives to MD simulation. a1 change in amplification only makes a difference from 10^(-4), and we initially thought that this difference would be difficult to achieve. However, since \Delta (\Delta G) is -5.67 kcal/mol, the professor said that this is a value that is not impossible to achieve with only a single nucleotide mismatch. Until now, we have only focused on how many free energies are available in the perfect-matched case. However, he pointed out that we need to consider how much the free energy changes when single-mismatch is introduced, and whether we can distinguish SNPs by that.

Since it is difficult to obtain the reaction rate constant, he suggested that it would be better to use the NN model to explain the difference in specificity. However, he pointed out that the current model is not enough to show the difference in specificity between EXPAR and TWJ, so it is necessary to create a new model. An example is that the binding of target and helper to template is done separately. The advantage of TWJ is that the reaction stops when there is a mismatch because the binding reaction is done little by little. If a reaction in which individual sequences (target and helper) are attached to the template is added in between, the structure will be unstable and the reverse reaction will not be negligible, which may make a difference.

For now, what we can do is to check if the equilibrium constant will never be 10^(-4) using the NN model, and then it would be suggested that it could be as low as 10^(-3). He also said that we need to make a phase diagram of the yield of the final product with ln (k1) and ln (a1) on the horizontal and vertical axes, respectively.
He also pointed out that we need to check the database to see how many sequences in the tear fluid are close to the biomarkers. He said that if there are no SNPs, it would be good to be able to show that TWJ is more specific than EXPAR, even if we cannot prove the result that TWJ can also discriminate against SNPs. But if we can get a good result, we can say that TWJ is robust against SNPs.

Finally, he gave us Maslow's quote, "If the only tool you have is a hammer, everything looks like a nail."

Team Reflection

It turned out to be good to evaluate the difference in specificity between TWJ and EXPAR by the difference in free energy, as MD of RNA-DNA is difficult. However, the current model does not show any difference in energy, so we need to create a new model and decided to consider consulting with a sequence design expert. We need to find out if there are sequences in the tear fluid that are close to the biomarker, and work backwards from the goal to determine how much specificity is needed.

Dialogs with Prof. Toru Terada

Affiliation: Professor, Department of Applied Bioengineering, Graduate School of Agricultural and Life Sciences, The University of Tokyo

Planning to evaluate the stability of the Cascade-crRNA complex by performing MD simulations of the interaction between Cas and nucleic acids, we decided to have a conversation with Prof. Terada, who performs MD simulations of biomolecules.

Discussions

First, we explained our plan to investigate the stability of the complex between Cascade and crRNA, based on a past iGEM team in China. Since the sorts of protein and crRNA are different from previous research, we needed to change the initial crystal structure for simulations. As for parameter source, he advised us to use our experimental data for the protein.
Also, he explained that RNA can be predicted sufficiently using software such as Alpha Fold. In general, larger systems lead to complex simulation and longer time to get stable results, but he said that the size of Cas3 would not cause too much trouble if we adjust the simulation time. Regarding the simulation time, 20 ns, which we were planning, is short in the research field, but can be considered to be a reasonable simulation time against a short period until Jamboree, based on his advice.

We learned that Gaussian accelerated molecular dynamics is used in MD simulations to investigate off-target effects, but it is difficult to adjust the range of potential energy. He advised us that while it is good to refer to the paper if there is a description of how to adjust the parameters, it may be faster to conduct experiments in Wet lab to search for parameters that show stable behavior.

Furthermore, we asked about a kind of MD simulation useful to investigate whether unintended reactions would inhibit the target reaction when gold nanoparticles are attached to the end of dsDNA. He said that the presence or absence of interactions between gold nanoparticles and other molecules is important. Specifically, if there is no interaction MD simulation is not necessary even if the gold nanoparticle force field exists, and to perform MD simulation,bindings between gold nanoparticles and DNA must be made explicit.

Team Reflection

Our project plan was basically approved. We realized that the accuracy and cost are greatly affected by the simulation time. As the first step, we decided to analyze the stability of the Cascade and crRNA complex using MD simulation, and then tackle the rest tasks with remaining time.

Dialogue with Prof. Kaori Muto

Affiliation: Professor, Department of Public Policy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

We were refining our project ideas with the goal of rapid societal implementation. We believed that by avoiding Japan's Cartagena Law, we could expedite the implementation process, and we considered that our cell-free system avoided the application of the Cartagena Law
To confirm whether this idea truly avoids the Cartagena Law, we consulted Prof. Muto, an expert in the field of public policy.

Interview

Since our research involves human subjects, Prof. Muto informed us that we are required to submit an ethics application, as it falls under the national ethical guidelines. Regarding the method of tear fluid collection, it was suggested that it might be classified as minimally invasive. Prof. Muto also mentioned that we may be able to obtain tear fluid from patient associations, as many people are likely willing to cooperate.

As for the Cartagena Law, Prof. Muto confirmed that our current idea does avoid it, as presumed in our initial plan. However, she also noted that if a larger number of people begin using this system, it’s uncertain whether the project will remain entirely outside the scope of the Cartagena Law.

We hadn't previously considered the dual-use nature of the project, but during the interview, we discussed dual-use concerns and safety. While potential false positives and the psychological burden of discovering a glaucoma risk were considered, no military dual-use was identified at this stage.
Nonetheless, we recognize that as scientists, we have a responsibility to always consider the possibility of abuse by third parties. Additionally, Prof. Muto pointed out that since users will directly see the results, there’s a possibility of increased psychological burden or misunderstanding.

The Cartagena Law approved in Japan has not been well integrated with the Pharmaceutical Affairs Law, which is considered a factor in the delayed development of synthetic biology in Japan. We believe that if we can approach synthetic biology not only from a scientific standpoint but also from a more political perspective, this delay can be addressed.

Team Reflection

Based on our discussion with Prof. Muto, we decided to proceed with submitting the ethics application. Additionally, we were informed that at this stage, the project avoids the scope of the Cartagena Law. For the time being, we have shifted our focus away from heavily considering the Cartagena Law and will move forward with the project.

Furthermore, while no dual-use potential for military purposes has been identified at this point, we, as scientists, believe it is our responsibility to always consider the potential for misuse by third parties.
We also believe that by approaching synthetic biology not only from a scientific perspective but also from a more political angle, we can help bridge the development gap that currently exists between Japan and other countries.

Dialog with an anonymous lawyer

Since our project includes design of a device, we would have liked to learn more about the laws and regulations on commercialization. Based on our conversation with Dr. Shimizu in OUI Inc., we also wanted to ask a lawyer about appropriate places to conduct lobbying activities. We decided to interview a lawyer who is knowledgeable about the Pharmaceutical and Medical Device Act.

Interview

First, he explained that since the purpose is glaucoma detecting, our device is an IVD product and therefore regulated by the Pharmaceuticals and Medical Devices Act. According to that law, approval from the Ministry of Health, Labor and Welfare is basically required for the release of medicines and products. Regarding the place of lobbying activities, we should first consider getting permission from the Ministry of Health, Labor and Welfare, in order to advertise and sell.

Also, he explained about the roadmap for IVD products: first, PMDA (Pharmaceuticals and medical Devices Agency) will conduct a scientific review, and then the Ministry of Health, Labor and Welfare will consider approval.

He also introduced the Act against Unjustifiable Premiums and Misleading Representations in regards to advertising. Products must not be advertised in a way that gives false information or misunderstands.
We learned that there is no particular problem with obtaining feedback from buyers after selling a product. However, regarding the handling of the collected feedback, information on human health should be treated as confidential information and should not be leaked to the outside. The acquisition itself is not a problem, but care must be taken after acquisition.

Next, our topic moved on to the approval of IVD products. He explained that IVD products are relatively quickly approved if there is enough data. We were advised to ask the PMDA for the necessary data and product details.

We were also informed that there is no problem with customers’ troubles with the products, as long as they are properly described in the instruction manual. In this regard, liability arises only when it is shown that there is a defect in the product that caused the accident, and according to the Product Liability Act, companies are not held responsible when problems arise from unexpected use such as drug overdoses.

Finally, we knew that PMDA requirements are dependent on the invasiveness of the product, and that applications must be submitted and approved for each product for each disease.

Team Reflection

Through this discussion, we learned many important things about products for sale. Our detection device is an IVD product and is regulated by the Pharmaceuticals and Medical Devices Act. It can be advertised and sold only after the Ministry of Health, Labor and Welfare issues a permission after scientific review by the PMDA. It is important to be careful not to make false or misleading advertisements when advertising. In addition, there is no problem with unexpected situations as long as they are properly described in the instruction manual. Furthermore, the requirements imposed by the PMDA vary depending on the invasiveness of the sample collection.

After this interview, we decided to go to the PMDA after the technical aspects were firmly established. Furthermore, we believe that we have a responsibility to improve our products by obtaining feedback from purchasers after sales. We will be definitely sure that care must be taken when handling data.

Dialogue with C4U

C4U is a startup from Mashimo Lab.
We conducted an interview with C4U to learn more about their efforts to gain market share in a very broad market, their management strategy, and their quality control methods.

Interview

Regarding strategies for gaining market share in a broad market, it was explained that the approach differs depending on whether it's B2C or B2B, so it should be decided based on the sales format. For a wide market, approaching consulting firms was suggested, while for pharmaceutical approval, a scientific approach was recommended.
It was mentioned that for health check-ups, promotion could be sufficient without pharmaceutical approval.

Concerning quality control efforts for stable results at an industrial production level, it was noted that large-scale supply requires different techniques from lab-level technology, so they generally view this negatively. If manufacturing isn't the primary focus, outsourcing was suggested as a wise choice. Once large-scale manufacturing facilities are established, there's no need to keep them in-house.

When speaking to the general public, it was emphasized that communicating the benefits of using the product is crucial.

It was also pointed out that to obtain pharmaceutical approval, it's necessary to demonstrate positive impacts not just for patients, but for the entire healthcare system. While price simulations are important, it was noted that reagent costs alone can't determine product pricing, and factors like freeze-drying costs and manufacturing techniques should be considered.
It was explained that the cost of existing fundus examinations is low and considered cheaper than devices, and it was pointed out whether there is any chance of success. It was suggested that the need for this device should be clearly justified, considering that if precise examinations are needed after using the device, why not just do the precise examination from the start.

It was also mentioned that markets for cancer or STD detections might be larger and require more frequent detecting than glaucoma, and made some suggestions for application in this direction.
The importance of setting a cut-off value for the detection was emphasized, despite limited data. It was explained that this value would differ based on whether false positives or false negatives are prioritized, and a strategy should be developed accordingly. Lastly, it was suggested that using smartphone cameras and AI to analyze LFA image color intensity might be more accurate.

Team Reflection

Large-scale production requires different technologies than those used at the laboratory level. Therefore, our best strategy is to refine our laboratory-level technology and then outsource industrial production to external partners.

Regarding pricing, we received quite severe feedback. We need to calculate an appropriate price and identify significant benefits of using POIROT compared to undergoing cheaper, more precise examinations.

Concerning false positive and false negative rates, if the goal is early detection, we must identify groups where expression levels have increased but are not yet very high. Given the nature of glaucoma, it would be problematic if our detection device caused more people to avoid visiting an eye doctor due to negative results. Therefore, we believe that strategically, it might be better to reduce the false negative rate at the cost of increasing the false positive rate.

Dialogue with Dr. Michio Suzuki

Affiliation: Professor, Department of Applied Life Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo

We are considering using the Lateral flow assay proposed by Dr. Wakui and Prof. Mashimo as a final device for displaying results, and gold nanoparticles will be used in this process.
Industrial production of gold nanoparticles requires high temperature and high pressure, which has a severe environmental impact. We interviewed Dr. Suzuki, who has been creating gold nanoparticles using lactic acid bacteria.

Interview

He explained that lactic acid bacteria can not only create gold nanoparticles specifically, but also decompose other heavy metals as a type of bioremediation.
He said that a glycoprotein on the cell membrane called DGDG is the key to the preparation of gold nanoparticles using lactic acid bacteria. DGDG of the bacteria reduces gold, which is then incorporated into the cell surface and inside the cell to produce gold nanoparticles.

Since our laboratory mainly deals with E. coli, we asked him about E. coli, but he said that it is a little difficult to do so with E. coli. He tried to make DGDG in E. coli, but the particle size is larger than that of gold nanoparticles made from lactic acid bacteria, the culture conditions are difficult, and E. coli is likely to die during the culture process.

Compared to the existing method of creating gold nanoparticles using citric acid, it is inferior in terms of cost, but he explained that the major advantage is that it is done at room temperature and pressure and does not use organic solvents. In addition to gold, other metals such as lead can also be used to create nanoparticles, he said. Possible applications include scattering them in the soil and removing metals from the soil in collaboration with plants, and creating plants that can easily take in metals by incorporating a gene that produces DGDG into plants.

When the gold nanoparticles are removed from the microorganisms, if they are not genetically modified, they need to be shaken off by centrifugation, and if they are genetically modified, they need to be autoclaved to prevent them from spreading into the environment.

Team Reflection

In order to reduce the environmental impact of the entire system, we asked how to create the gold nanoparticles used in the Lateral flow assay. At this time, it is not competitive with existing methods in terms of cost, but has significant advantages in terms of energy and environmental pollution. For the convenience of our lab, we would like to use E. coli, but he said he already has more data on lactic acid bacteria, and it is easier to start experiments with.
We decided to consider whether to actually conduct the experiment based on the progress of the isothermal amplification system experiment, while also considering the possibility of starting a new experiment to create gold nanoparticles with E. coli.

Dialogue with Hemicellulose

Hemicellulose is a biodegradable plastic company.
We cared about the exterior of POIROT, and would have liked to know if there was a biodegradable plastic that was environmentally friendly and easy to use and what the price would be.

Interview

First, we found that there would be no biodegradable plastic that meets the strict requirements to withstand RNase sterilization, which could be used for laboratory equipment. On the other hand, there are biomass-based plastics that are not biodegradable. Considering that all of the solutions inside POIROT are disposed of as combustible waste at home, it is desirable for the exterior to be biodegradable as well. Also, as another solution, we were informed that sterilizing spray, such as RNase free spray would be available. The spray has a pH of 11 and can be used on some biodegradable plastics, and it costs about 1,000 to several thousand yen per kg.

We also learned that strict sterilization like what we had in mind was not necessary even for the case of influenza or COVID-19. Since we were requiring sterilization that is stricter than medical sterilization, he pointed out that we needed to think about the level of sterilization required.

As for the creation of the exterior, molding can be done with a 3D printer. It can be made from one piece, and can be mass-produced by making a mold. For mass production, it costs about 1 to 2 million yen and the delivery time is more than one month. Once a mold is prepared, making 1,000 pieces in a day is possible.

Team Reflection

After talking with Hemicellulose, we started considering the idea of ​​making the device using biodegradable plastic sterilized with RNase free spray. The advantage of using biodegradable plastic for the exterior is that it can be thrown away together with the contents in the burnable garbage after being used at home. This is in response to the people' requests for products that are easy to dispose of and environmentally friendly. At this point, we should not compromise.
Click here for the Entrepreneurship page for future plans such as pricing

Dialogue with Glaucoma Friend Network (GFN)

Glaucoma patient organizations.

Glaucoma Friend Network (GFN) is a membership organization run by and for glaucoma patients. Their activities are of wide variety, including study sessions on glaucoma, the publication of newsletters, encouraging interactions among members, and raising public awareness of the significance of early detection of glaucoma.
Through the dialogue with GFN, we aimed to hear opinions from patients about the glaucoma detection and the importance of early detection.

Interview

The members of GFN showed strong interest in our activities. They not only provided us with valuable feedback on our project but also shared their concerns and the burdens they experience regarding their treatment as patients.
Firstly, as for the feedback on the glaucoma detection device for household use itself, concerns were raised about the possibility that users might mistakenly assume they are fine with a false negative result.
They shared with us that many patients, after being diagnosed with glaucoma, panic after searching on Google about glaucoma symptoms or become depressed due to the fear of going blind. This has even led some to quit their jobs or suffer from significant emotional stress. They emphasized the importance of telling that early detection can help slow the progression of the disease, to prevent such misunderstandings.
From these points, since glaucoma often presents no noticeable symptoms in the early stages, they suggested that a detection device we propose could potentially reduce psychological anxiety.
They also mentioned that the fact that glaucoma diagnosis needs an amount of time makes frequent hospital visits burdensome. Moreover, even after being diagnosed with glaucoma, patients often need to apply multiple types of eye drops, which can be quite tedious. Due to such burdens and the lack of obvious symptoms until the condition progresses, some people stop attending their appointments on their own, highlighting the potential of our device to raise awareness and create a sense of urgency for those individuals.
We asked the two interviewees about the progression of their glaucoma. One of them told us that they were in the mid-stage of glaucoma when they first visited an eye doctor, and the progression remarkably slowed after starting the treatment. They said that they can still look at the computer screen all day without issues, but acknowledged that some people cannot manage tiredness in their eyes.
The other interviewee, whose condition has progressed much further, told us that he can no longer read books or newspapers, and that watching TV is difficult without audio assistance; His eyes cannot keep tracking movements and feeling exhausted. He mentioned that he does not watch so much as “listens to" TVs, almost only NHK programs, especially dramas in the morning. He also mentioned that due to the loss of 70% of vision in the left eye and 30% in the right eye, he uses a white cane. However, he said that steps are frightening, he can't see traffic lights, and he's had several near-miss accidents. When he goes to eat at a restaurant, he can no longer read recommended menus handwritten on the blackboard and can only rely on the standard menus at chain restaurants. He also mentioned that when ordering a new menu item, he sometimes mistakes tomatoes for pickled plums. Furthermore, it's difficult for him to distinguish people's faces, and sometimes he can’t tell someone's gender.

We asked what is necessary to change such situations.

The interviewees from GFN emphasized that glaucoma is hard to detect without an eye exam at an eye doctor. Based on that, they suggested that glaucoma detecting should be made mandatory in corporate health checkups or municipal health screenings, even though it is not currently required. They pointed out that even in comprehensive medical checkups, the results are reviewed by an internist rather than an eye doctor in some cases, meaning that aspects related to internal medicine are prioritized, and glaucoma can easily be overlooked. They stressed the importance of incorporating glaucoma detecting into health screenings and ensuring people capable of picking up on the results afterward.
Furthermore, they emphasized the need to create detecting devices that can be used easily, even by elderly individuals who may have difficulty with hand movements, especially considering that many glaucoma patients are older.

When asked about awareness activities for early detection, GFN members strongly emphasized the importance of early detection, which they only realized after the disease had progressed.
One such awareness activity is the "Light Up Green" campaign by the Glaucoma Society which aims to raise awareness before it's too late for treatment. GFN also participates in the Light Up Green campaign. They mentioned that they would appreciate proposals from young students like us. Additionally, GFN is involved in various activities, such as conducting awareness campaigns through online broadcasts. However, they pointed out that creating videos to reach a wide audience remains a challenge.

They pointed out that modern society is saturated with information, and young people have too many things to take care of, leaving little room to think about glaucoma. Even if they are told in the early stages that they show signs of glaucoma, it often gets pushed down their priority list. GFN members mentioned that they had done the same. They emphasized the importance of creating criteria to raise the priority of glaucoma and reiterated the necessity of our project.

They also raised the issue of treatment continuation, which is another challenge, even after early diagnosis. They noted that after starting eye drops and lowering intraocular pressure, many people do not feel a significant change and tend to drop out of treatment midway. "A year after diagnosis, many patients have already stopped treatment. Those in their primes think that it’s not changing much and let it progress... Meanwhile, the optic nerve damage that occurs cannot be recovered," they said. This highlights the importance of both early diagnosis and treatment continuation.

On a social level, they mentioned that in any country or city, the dependence on computers will continue to rise, and eventually, even cash may disappear. In such a society, it is easy to imagine that the strain on our eyes will increase significantly. They believe the incidence of glaucoma will rise, and it might even affect younger people and children. For this reason, they emphasized that tools like the proposed screening kits are crucial and could become widely used within 5 to 10 years.

Lastly, we were able to ask about the visual field defects experienced by GFN members. They noted that the way vision is affected varies from person to person. As a reference, they kindly provided us with images showing visual field defects. We are truly grateful for their cooperation.

Team Reflection

After interviewing GFN, we realized that our approach is both highly beneficial and meaningful. We also learned about the importance of early detection and the mental burden faced by individuals diagnosed with glaucoma. It is essential for us to raise awareness on this issue. For more details on our approach, please refer to the Education section.
Furthermore, they highlighted the potential of our project to reduce the mental burden on patients, as well as the concern that some individuals may discontinue their visits based on personal judgment. This is an important point to consider for future applications.

Dialog with TODAI TLO
(technology licensing organization)

TODAI TLO is a subsidiary of the University of Tokyo that handles intellectual property and patents.
We interviewed TLO to find out what steps need to be taken to turn POIROT's technology into intellectual property.

Interview

We recognized that our project, which combines existing methods, is a new invention, and asked her whether our system could obtain a patent by filing an invention notification. In response to this question, we received an answer that the ingenuity used to combine existing methods itself falls within the scope of the patent. We think that the ingenuity in our case is the Multistep sequence and assembly method. She said that it is possible for us to file an application at that time, but it may be difficult to obtain a wide scope of rights.
To file an application, she said, the focus of our invention is whether it is actually possible to make an accurate judgment with tear fluid using POIROT.

On the other hand, our data was evaluated to file an invention notification to some extent. She also told us that some questions may come from TLOs and patent attorneys about data that is missing when filing an application.
Furthermore, we learned that even if an invention notification is rejected once, we can revise and resubmit. However, it is smoother to submit it when it is really necessary. In addition, we were informed that applications cannot be made within one year of announcement.

When trying to obtain patents for limited sequences such as genes and proteins, there are often other completed products already in existence. Patents are valuable for the purpose of protecting a company's products, but not for developing or selling its own products. It is often difficult to apply for a patent with detailed conditions such as sequence restrictions. In this case, it seems less beneficial for universities which will file patent applications with the aim of licensing to companies. On the other hand, if someone starts their venture company and has a clear goal of commercialization, it may be meaningful to apply for a patent with only a limited sequence.

Students can choose to apply for a patent individually or through the university. If the application is through the university, it will be made at the university's expense, and the university will maintain and manage the patent application. In addition, if the university has entered into a license agreement with a company, they can file an objection if infringement occurs, depending on the company's opinions.

Team Reflection

We now know that the focus of our patent is whether POIROT can actually make a judgment using tear fluid, so we need to continue our experiments and demonstrate that glaucoma can indeed be detected using tear fluid. In addition, patents will be necessary if thinking of starting a startup in the future.
Finally, at the end of this interview, she introduced us to an incubator to help with thinking about a business model.

Dialog with AIZOTH

Mr. Kawajiri is the CEO of AIZOTH Inc. and specializes in analyzing experimental data using AI.
We visited him to ask about the possibility of adopting AI to develop our project.

Interview

Mr. Kawajiri was very interested in our project and expressed high hopes for it. He explained that they were developing a service in which AI analyzes experimental parameters and performs causal analysis and optimization of phenomena. The analysis software has a linkage analysis function, which is considered to be a good match for our project. We also heard that one of the strengths of the software is its ability to analyze even small amounts of data.
Unfortunately, we did not have enough time to adopt it before the Jamboree, but we could see the possibility of us working together to publish a paper in the future.

Team Reflection

Our project has the potential to develop significantly when combined with a variety of technologies.
Fortunately, Mr. Kawajiri has highly evaluated our project, so there is a possibility that he will provide significant support as we move beyond iGEM and look to publishing our findings in a journal article.