Entrepreneurship

Overview

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POIROT is a glaucoma testing device that can be quickly implemented in society. We intend POIROT to be used correctly in society, being a safe, reliable and commercially sustainable product. By carefully building a scenario to start a business that allows POIROT to withstand the stress of starting a business, we will explore the possibility of social implementation.

For developing the scenario, Entrepreneur was divided into two sections: asset recognition and growth strategy. Our first priority was to recognize the strengths and weaknesses of POIROT while understanding the situations of the demand side and the supply side. Based on this recognition, we established a business growth strategy that paved the way for commercialization.

Asset recognition

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Analyzing characteristics of a product helps us understand its strengths and weaknesses, current unmet needs, potential risks, and future growth potential. Understanding your current position and envisioning your future direction makes it possible to develop strategies to deal with the stress of starting a business. Namely, the first thing you need to do when developing a business startup scenario is to recognize your assets.

We analyze POIROT from two viewpoints: demand and supply. On the demand side, we will analyze the current market, its room for expansion, competition, and how much value customers see in POIROT. On the supply side, analyze POIROT's manufacturing and supply capabilities and value chain.

Demand

Analysis on the demand side.

People's needs and current market

Glaucoma is an eye disease that can affect anyone, and patients exist all over the world. The number of patients is estimated to be 80 million in 2020, and is predicted to rise to 110 million by 2040 ¹. Our country Japan is also suffering from this worldwide eye disease. The number of glaucoma patients in Japan is estimated to be approximately 2.3 million in 2020 ². The number of patients is said to be continuing to increase.

In Japan, a glaucoma epidemiological survey (Tajimi Study) conducted between 2000 and 2002 found that the new incidence of glaucoma was 89% ³, and the fact has revealed that a large number of people have glaucoma but have not been diagnosed and have not started treatment. In the treatment of glaucoma, early detection is important to delay the progression of symptoms through early treatment. However, many people in Japan have not been diagnosed with unnoticed symptoms. Early diagnosis before they become serious is imperative.

According to a 2007 study, the total economic cost of visual impairment in Japan is estimated to be approximately 3 trillion yen ⁴. Based on the percentage of people with visual impairments caused by glaucoma, a simple calculation results in an economic loss of approximately 750 billion yen per year.

Despite this critical and urgent need, the market for aiding in the early diagnosis of glaucoma is still in its infancy. There are some startup companies in Japan such as OUI inc., which performs remote ophthalmological diagnosis using smartphone cameras ⁵, and DeepEyeVision, which assists diagnosis of glaucoma from fundus images using deep learning ⁶, though there is no established business that contributes to the diagnosis of glaucoma using wet technology. POIROT can meet this untapped need, using wet technology to easily detect glaucoma at home.

• Technology

POIROT amplifies and quantifies glaucoma biomarker miRNA in tear fluid. Japan has ratified the Cartagena Convention ⁷enforces the Cartagena law ⁸. As a result, there are significant obstacles to removing genetically modified cells from the laboratory, hindering rapid social implementation. However, POIROT is an enzyme- and nucleic acid-based sensing device that does not use genetically modified cells and is therefore outside the scope of the Cartagena Act. In addition, it has high applicability, as it can be used not only for glaucoma but also for the detection of diseases that have disease-specific nucleic acid biomarkers. POIROT was not aimed for comprehensively searching for biomarkers, so it is not suitable for comprehensively searching for new biomarkers. POIROT's strengths are the fact that it can contribute to the early diagnosis of diseases other than glaucoma, such as cancer, which has been gaining attention in recent years. Wide range of commercial activities are expected to take advantage of POIROT's high flexibility, but we will first limit the application to the initial goal of early detection of glaucoma and recognize the market size of POIROT.

• TAM

TAM (Total Addressable Market) refers to the maximum market size that a business can achieve ⁹. In other words, it represents the total revenue that could be generated if all market shares of the glaucoma detection were acquired. Here, we assume that POIROT can detect all types of glaucoma other than acute glaucoma. According to the statistical surveys conducted by the Japanese government, the estimated number of outpatients who visited for the diagnosis of diseases related to the eye and adnexa was 358,000 ¹⁰. It is said that a single glaucoma diagnosis, including tests such as ophthalmoscopy and visual field tests, costs patients about 4,500 yen ¹¹ (though the cost is reduced by the general Japanese healthcare insurance system). Assuming that patients go see their eye doctor three times a year, as it is recommended once in three to six months, we can estimate that the size of the glaucoma detection market in Japan is approximately 5 billion yen

In addition, in Japan, workers are required to undergo a health checkup once a year, and many people undergo the medical checkup. Though a medical checkup is where the potential for glaucoma can be indicated, ophthalmoscopy is excluded from the mandatory checkup items in the current medical checkup. Here, we assume that a medical checkup is included in the glaucoma detection market. The population over 40 years old is 78 million ¹², and we also assume that all the population undergoes the medical checkup once a year. Although there is no uniform cost for a medical checkup, we suppose the glaucoma detections in a medical checkup are similar to those of the hospitals. From these assumptions, the market size can be estimated to be 1 trillion yen. The glaucoma detection market can be considered to be stable, since anyone can suffer from glaucoma. To further expand this market, it is necessary to emphasize the significance of glaucoma detection more widely and strongly, and to extend access to such detections to remote areas.

• SAM

SAM (Serviceable Available Market) refers to the maximum market size that can be achieved within the framework of TAM that POIROT can actually address ⁹. Not everyone who undergoes glaucoma detection voluntarily or passively through a medical checkup can be reached by POIROT. There are those who prefer direct diagnoses from ophthalmologists, who lack ophthalmoscopy in medical checkups, and who are unwilling to use services unfamiliar to them. To reach those who go see an eye doctor, it is essential to first inform the eye doctor about the benefits of POIROT. At the same time, we aim to get POIROT included in items of medical checkups conducted by enterprises. By conveying the potential economic losses and risks associated with employees suffering from glaucoma to corporations, so that POIROT can be incorporated into medical checkups as a part of employee benefits.

• SOM

Serviceable Obtainable Market (SOM) refers to the market size of customers that POIROT can actually target ⁹. Our initial focus is introducing POIROT to individuals who go see eye doctor medical checkups conducted by large corporations with well-established employee benefits. Introducing POIROT to medical checkups will lead to stable income and data accumulation. By building a track record, we can demonstrate the reliability and safety of POIROT, allowing to reach those who feel reservations.

Half of those who go see an eye doctor amounts to 180,000. As of 2006, the number of employees in large Japanese corporations was 12 million ¹³, and it is assumed that such large corporations conduct medical checkups. Therefore, the market size is estimated to be approximately 150 billion yen.

Kano Model

The Kano model ¹⁴ is a methodology that categorizes quality elements that affect customer satisfaction and uses these features in product development. The Kano model helps us to understand customers' needs and requirements of a product. In iGEM, Valencia_UPV2018 ¹⁵ has adopted this methodology in the past, which was referred to as a basis for POIROT's analysis. The below is a description of how the survey was conducted with the methodology. This is written for POIROT, but it is a guide for analysis based on the Kano model.

1. Collecting User Feedback

The product was presented to the general public multiple times, gathering not only positive feedback but also negative opinions, questions, and suggestions for improvement related to the project Education. To truly gain users' understanding, we considered their perspective and continuously refined the product, rather than merely promoting it in a one-sided manner.

2. Conducting a Survey

First, information about the product and its quality elements was presented to the users. Next, to identify the required quality elements, we asked paired questions, which took the form of "How would you feel if the product had quality element A?" and "How would you feel if the product did not have quality element A?" While A remains the same, the questions address the element from both a positive and negative perspective. Users then rate their answers on a five-point scale. The actual survey used is as follows:

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3. Analyzing the Data

Responses from the survey were compiled into a table and analyzed to determine whether the elements are truly necessary. It is essential to improve the product's quality elements based on this analysis. By incorporating or enhancing the quality features desired by users and removing the ones that are not, it becomes possible to develop a product that truly meets user needs.

Implementation

We used the Kano Model to analyze the quality elements required for POIROT. At the iGEM Japan Meetup, we presented POIROT to iGEMers and researchers and sought feedback from them. In addition to the paired questions mentioned above, we asked about the respondents' age and their expected price of POIROT, used for setting the price. After the presentation, we conducted a survey using Google Forms and received 30 responses. Since we obtained a sufficient number of valid responses, we made analyses on POIROT using the Kano Model. The detailed analysis result is as follows:

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① The desired quality features of POIROT itself included not only the availability at home but also its low false positive rate. Additionally, due to growing environmental awareness, the ease of disposal was highly valued.
② About features beside POIROT itself, respondents highlighted the improvement of being able to use without additional equipment and ease of purchase. Based on this analysis, we conducted Human Practice to improve POIROT's exterior and sales method.

SWOT Analysis

We continue to analyze POIROT.
We can improve the quality of POIROT not only by stressing the users' perspectives but also by analyzing from our perspective. To achieve this, we conducted SWOT analysis ¹⁶. The SWOT analysis evaluates the four elements including the strengths, weaknesses, opportunities, and threats in glaucoma detection, to accurately grasp the current situation regarding POIROT. In addition to the SWOT analysis within our team, we engaged in discussions with junior high and high school students and university students as a part of Education activities, taking in diverse perspectives.

Many iGEM projects perform SWOT analysis and just list features, but do not devise creative strategies based on the results of the analysis and reflect them in the project, which is the most significant part in SWOT analysis ¹⁷. The SWOT analysis should not be used merely to enhance the project's appearance but should be employed for truly responsible social implementation. How can we leverage the strengths? How can we overcome the weaknesses? How can we take advantage of the opportunities? How can we deal with threats and defend ourselves?

It is necessary to develop strategies for advancing the project based on these four elements. We used this SWOT analysis to consider methods for improving the project and to formulate strategies. It is crucial to develop strategies to advance the project based on the four elements. We used the SWOT analysis to consider methods for improving the project and to formulate strategies.

Extensibility

Including POIROT, we learned through Human Practice with OUI Inc. that it is vital to operate continuously when we start a business. To do so, it is necessary not only to obtain patents for our products to protect technology but also to improve our products and to explore new business opportunities. The future business strategy will be explained in the second section. Here, we will discuss the scalability based on the analysis of Kano Model and SWOT.

Extensibility of POIROT

POIROT is developed as a tool to assist the early diagnosis of glaucoma. However, the flexibility of POIROT is not limited to glaucoma. By simply altering the sequence of the region that hybridizes with the target miRNA, it can be used to detect various miRNAs without making a substantial change in the entire sequence. Provided that disease-specific biomarker miRNAs are reported, diseases based on various liquid samples, including tear fluids.

For example, it can greatly contribute to the early detection of diseases that are challenging to identify in their initial stages, such as glaucoma, as well as diseases like pancreatic cancer, which are difficult to detect with current methods but remain a top priority for government health initiatives ¹⁸.

Utilizing the quantitativeness of POIROT enables tracking disease progression. If biomarker miRNAs that reflect the progression of a disease exist, continuous use of POIROT allows for ongoing monitoring of the condition. For example, it may enable tracking of diseases requiring close attention to progression, such as chronic conditions, or monitoring treatment outcomes from nucleic acid-based therapies, which have gained attention in recent years, from home. A potential model involves users measuring concentrations with POIROT, sending the results to the hospital, and having doctors evaluate the effectiveness of the treatment. This approach could reduce the physical and time burdens on patients and be implemented even in remote areas.

While POIROT has the potential to expand into markets beyond glaucoma, such as cancer and infectious disease detection, it relies on the assumption that disease-specific nucleic acid biomarkers exist for each. POIROT is not well-suited for comprehensive biomarker searching, so identifying new biomarkers is more efficiently done with existing methods ¹⁹. In other words, the current biomarker data might be insufficient for detecting neglected diseases or novel infectious diseases, making POIROT less optimal. To develop POIROT, it is necessary to support biomarker search.

Extensibility around POIROT

POIROT is specialized in miRNA detection. Its detection performance is the same or exceeds the existing systems, and to further enhance the quality expansion of associated devices is necessary.

The risks of tear collecting in the POIROT were pointed out by Human Practiceto eye doctors and POCT experts. A conventional method of using Schirmer test paper to collect tears, which we were initially planning to adopt, is a familiar method for eye doctors, but not for general users who are not healthcare professionals. Though minimally invasive, placing Schirmer test paper on the eyelid for several minutes could potentially lead to damage to the eye. Moreover, someone with malicious intent may contaminate Schirmer test paper, leading to the pathogen intrusion into the eye. Surely, such health risks could negatively impact the business itself, and we need to reduce the risks in developing a product that is responsible for users' safety.

We discussed alternative methods for collecting tear fluid in place of Shrimer test paper in the HP. Dr. Inagaki, an eye doctor, introduced us to strip meniscometry ²⁰. Using this method allows collecting tears more quickly and effectively than using Shrimer test paper, because in this method, tears can be collected by immersing a strip in the tear meniscus for 5 seconds. Additionally, in the discussion with OUT inc., the possibility of using eye wash solution was suggested. Since his method does not require bringing paper close to eyes, the risk of foreign objects coming into contact with the eye is eliminated. By washing away the tears containing miRNAs at the surface of the eyes using the solution and using the collected solution for detection, it becomes possible to collect tears non-invasively.

To reflect the high awareness towards environmental problems of users proven by the Kano model, we held HP with Hemicellulose, which is a corporation involved in the development and processing of biodegradable plastics. All the reagents used in POIROT are of composition and concentration that pose no harm to the environment, and therefore it can be disposed of as a combustible waste. To take advantage of the benefit that it can be thrown away as it is, we considered making outer casing of plastics so that it can be discarded as a combustible waste. The cost will be discussed later. POIROT requires ensuring not to degrade miRNAs, and we were also introduced to several biodegradable plastics that can withstand RNase Free processing ²¹. By adopting these methods, the entire POIROT can be thrown away as a combustible waste.

Next, we try to enhance services other than the product itself. While POIROT can be used for glaucoma detection, it cannot treat glaucoma. It is necessary to develop a system that allows users to smoothly access treatment. Speaking of glaucoma, not only early detection but also continuous treatment is crucial, which requires regular visits to eye doctors.We will create a web system that introduces us to nearby eye clinics from where the user is. The system enables users to make online appointments and send the results obtained from POIROT to eye doctors. Moreover, by forming partnership services from other corporations that conduct ophthalmoscopy and building a system that enables remote testing and diagnosis for glaucoma, thereby improving convenience for users.partnering with other companies offering fundus examination services, we aim to build a system that enables remote testing and diagnosis for glaucoma, thereby improving convenience.

Competition

A monopoly by a single product does not exist in a free market, as there will inevitably be competing products from other corporations. It is rare for a single factor to be the solution in addressing and improving social issues. In this section, we investigate the presence of direct, indirect, and potential future competitors to POIROT and analyze their impact on the future of POIROT.

A direct competitor refers to products or services that are similar or are of the same market. Here, the examples are glaucoma detection and cancer detection. The following are the list of businesses involved:

An indirect competitor refers to products or services different from POIROT but are targeting similar audiences.

Future competitors refer to those that may emerge in the future. There is the potential for groundbreaking methods in glaucoma diagnosis or treatments aimed at complete cures to appear. This also includes possible future improvements related to POIROT, such as the development of non-invasive methods for tear collection.

Competitors are not merely obstacles to the commercialization of POIROT but can also aid in glaucoma detection and other advancements. While sustaining the business is important when POIROT is launched, addressing the broader issues surrounding glaucoma and cancer is of greater significance to society as a whole. Although profit maximization is a fundamental principle in a capitalist economy, being mindful of social responsibility is also a core value for scientists.

Supply

Next, we will analyze the supply side.

Manufacturing and supply

What must be addressed when commercializing the product is the scaling up of manufacturing and supply capacity. Just as different views can be seen from different perspectives, there is a great possibility that what was possible at the laboratory level will become difficult or face new problems when brought to the industrial level. Since we, who have never been involved in actual manufacturing, could not fully discuss the problem, we do Human Practice with C4U, which is a social implementation using CRISPR and highlighted the problem. We analyzed the points that need to be improved when scaling up the manufacturing supply capacity to the industrial level.

1. Quality

One of the most important things consumers look for when purchasing a product is quality. Consumers demand reliability in disease testing products, even if they are not perfect. If a company cannot provide a reliable product even after scaling up to the industrial level, the product will not be purchased and the business may be scrapped as a result.

The amplification system used in POIROT has very good laboratory performance in terms of both sensitivity and specificity. Furthermore, some existing methods have even been reported to be able to detect miRNAs at concentrations several orders of magnitude lower than the miRNAs found in tear fluid ²². However, not everyone has laboratory-level equipment, techniques, and accumulated knowledge. Therefore, it is safe to say that it is impossible for consumers to reproduce the laboratory.Although there are excellent examples such as LAMP, which has been successfully developed into a kit ²³, the inability to reproduce laboratory-level performance is the reason why SDA-based POCT devices have yet to be implemented in society.

The following table summarizes the improvements that can be made to ensure that performance, such as sensitivity and specificity, is not compromised when manufactured at the industrial level.

Table 1: Qualty Improvement

Points of Improvement	Reason	Improvement Method
management method	Transport and sale of enzymes and nucleic acids while they are dissolved in Buffer may result in a decrease in activity. Moisture and other factors may thaw some of the enzymes and cause them to start moving.	Pay attention to storage methods. Protect contents using lyophilization techniques. Add appropriate protective agents to avoid loss of activity. Store in a manner that preserves the condition in which it was manufactured, avoiding high temperatures and high humidity. Design the device such that the inside of the device is not in contact with the outside so that the contents are not contaminated.
lacrimal fluid collection	Few laypersons have any experience in collecting tear fluid. There is a possibility that placing a Schirmer test strip over the eye may damage the eye in the unlikely event that it is not properly collected.	To enclose instructions on the use of Schirmer test papers. In addition, the company will post a video on its website regarding the proper use of the test papers to educate the public about the proper use of the test papers so that misuse of the test papers does not lead to anger. To use a less invasive method of lacrimal fluid collection than Schirmer test strips.
Sterilization of devices	The miRNAs in tear fluid are microscopic and there are many factors that degrade RNA in the general environment. Contamination inside the device may prevent correct amplification.	The most likely area for contamination is the exterior biodegradable plastic. To prevent RNase from being contained inside the device, it should be treated to be RNase free.



2. Cost

In selling a product, price is directly related to consumers' willingness to purchase it. If the price of a product is low, consumers are more likely to purchase it, but this leads to distrust of the product quality. Conversely, if the price is high, consumers are less likely to purchase it, but they will have more confidence in the product quality. If the price is set too low, the company will not be able to make a profit, leading to poor business performance. If the price is set too high, the product will not be purchased by consumers, leading to poor business performance. In other words, without setting appropriate prices, it will be difficult to continue as a business.

First, the cost of producing POIROT is calculated. The following cost is calculated based on the cost of purchasing reagents, etc. in the laboratory; it is the price for one lane; to use it for n different TARGETs, increase the number of lanes and multiply the total by n.

Table 2: Price list of contents

item	price (yen)
Schirmer test strip	8
synthetic oligonucleotide DNA/RNA	1
DNA polymerase	4
Nickase	1
dNTP	1
Cas3-Cascade/crRNA complex	300
total	615




Currently, it costs around 600 yen to detect a single target. While using multiple targets can improve detection accuracy, it also increases the cost. In addition to the contents of the device, there are also costs associated with creating the external casing. Through Human Practice with Hemicellulose Co., we explored the possibility of creating a biodegradable plastic. Excluding the cost of mold creation, though it depends on the type of plastic, it can be produced at a cost ranging from several hundred to several thousand yen per kilogram. Referring to the similar products ²⁴ with comparable shapes, approximately 10 grams per device would result in a production cost of about 10 yen per unit.

There are several types of profit, but here we will focus on the gross profit margin. The gross profit margin is calculated by dividing the gross profit by the sales and multiplying by 100. In Japan, pharmaceuticals are classified under the pharmaceutical manufacturing industry, and the gross profit margin for the manufacturing industry in 2021 was 21% ²⁵. It has been around 20% annually, so the goal for POIROT's gross profit margin is set at over 20%. To achieve this goal, the price for detecting a single target would need to be 720 yen.

Next, we investigated the desired price for POIROT. We conducted price surveys during Education activities and at Japan Meetups. The survey question was: "At what price would you consider purchasing a detection kit? In general, a glaucoma examination at an ophthalmology clinic in Japan costs about 4,500 yen, with 30% being the patient's responsibility."

Survey options:

1. Less than 500 yen
2. Between 500 yen and 1,000 yen
3. Between 1,000 yen and 1,500 yen
4. Between 1,500 yen and 2,000 yen
5. Between 2,000 yen and 2,500 yen
6. Between 2,500 yen and 3,000 yen
7. More than 3,000 yen



From these results, it was found that consumers prefer a price range of around 1,000 to 1,500 yen. Though the healthcare system helps reduce the burden of medical expenses, consumers expect a price for testing that is about one-third of the cost of a glaucoma examination at a hospital.

Based on the above results, we will set the sales price. Consumers desire a price range of around 1,000 to 1,500 yen, and we aim to improve detection accuracy by detecting multiple targets. Therefore, we will increase the number of lanes to detect two targets simultaneously and sell the product for 1,500 yen. At this price point, including the cost of the casing, POIROT's gross profit margin will be approximately 20%. This is about one-third of the cost of a glaucoma examination at a hospital and could help to prevent an increase in medical costs for the diagnosis.

To improve test accuracy while increasing the gross profit margin, it is necessary to reduce costs related to production and sales. However, calculating only the cost of reagents is insufficient to determine the actual product price. There are two major issues that affect the price of POIROT.

The first issue is temperature control for reagents. It is necessary to freeze-dry the reagents and seal them in the device to prevent reactions between enzymes and template DNA. Freeze-drying is required to ensure the reagents remain intact, which incurs additional costs. Moreover, the finished devices must be stored at 2-8°C, necessitating proper temperature control during storage and transportation, which adds further costs.

The second issue is the need to establish large-scale production technology for the Cas3-Cascade/crRNA complex. The creation of the Cas3-Cascade/crRNA complex involves the use of organisms such as E. coli or insect cells. Cultivation at the laboratory level differs significantly from cultivation at an industrial scale, with entirely different techniques required for culturing at 1 L compared to 100 L or 10,000 L. Once the cultivation technology is established, there is no longer a need to retain the personnel involved in its development. While outsourcing cultivation could resolve this issue, the costs associated with preparing for large-scale cultivation must still be recovered, which would inevitably increase the price of the device.

3. Delivery Time

By appropriately evaluating the annual shipment volume and the time it takes for products to reach the market, we can eliminate the spatial burden caused by accumulating inventory. There are still many uncertainties regarding product shelf life and durability, as there is potential for degradation over time. Therefore, forecasting supply based on long-term storage is a poor strategy. By achieving an appropriate supply relative to demand, we can provide consumers with the experience of having access to products when needed, thereby enhancing trust in both the products and the company.

First, we will estimate the delivery time for POIROT based on the procurement of reagents at the laboratory level. According to the Demand SOM, we need to establish a production and shipment system capable of serving approximately 12 million users annually. However, for the purposes of estimation, it is more realistic to handle a smaller figure, and we will consider the case of supplying 1,000 people. At this level, the demand for reagents is relatively low, and the time required for production is expected to be similar to standard timelines.

When considering the delivery time for POIROT, the most time-consuming aspect is the creation of the Cas3-Cascade/crRNA complex. Currently, production is primarily carried out by Nippon Gene, and the lead time from sequence design is estimated to be 3 to 4 months ^{26, 27}. During the period of creating the Cas3-Cascade/crRNA complex, it is feasible to prepare the necessary enzymes, nucleic acids, and packaging. The packaging will be made from biodegradable plastic. Selecting the resin and designing the mold will take over a month, but once the mold is completed, it will take less than a day to produce approximately 1,000 units. As soon as the Cas3-Cascade/crRNA complex is ready, the reagents will be mixed and freeze-dried. The freeze-drying process can take several dozen hours; here, we assume it will take 72 hours (3 days) to complete in-house ²⁸. Packing the final product will take approximately 3 to 4 months. If the production base is located in the Kanto region, Japan Post indicates that domestic shipping from Kanto takes about 3 days ²⁹. Therefore, the entire process from production to delivery can be completed in roughly 5 months.

Next, we need to consider a strategy for scaling up production to over 1,000 units. The main challenge regarding delivery time is the creation of the Cas3-Cascade/crRNA complex, which is both complex and requires 3 to 4 months to produce. The crRNA sequence used in the Cas3-Cascade/crRNA complex for POIROT is fixed, and there is potential for increased efficiency by increasing the production volume at once.

4. Fulse Detection

For products like POIROT that are used for testing and diagnosis, false positive and false negative rates are critical factors. If we can clearly establish a cut-off value, we can avoid such issues; however, due to the limited research on tear fluid and the miRNAs contained within it, it is currently impossible to define a solid cut-off value. Nevertheless, we need to establish a pseudo cut-off value for colorimetric detection using LFA, which will influence the product's characteristics depending on whether we prioritize minimizing false positives or false negatives.



When considering upregulated miRNAs, if we prioritize minimizing false positives, the cut-off value will be set high, as shown in Figure.6 left. However, this will result in many true positives being classified as negatives, increasing the false negative rate. Conversely, if we prioritize minimizing false negatives, the cut-off value will be set low, as shown in Figure.6 right. This will lead to many true negatives being classified as positives, thereby increasing the false positive rate.

Value Chain

Value chain refers to dividing a business into processes and analyzing what value each process generates in order to gain market advantage and improve business efficiency ³⁰.Past iGEM teams also analyzed their projects using value chain ^{31, 32.We analyze POIROT using the value chain to evaluate not only the economic rationale, but also the combined stress resistance to technological accumulation and various supply risks.While it is important to calculate costs using specific figures, there are many unknowns in the field related to healthcare, so we will only describe strategies.}

We begin by classifying the economic activities related to the product into two categories: main activities and supporting activities. We will enumerate what is included in each activity and analyze POIROT.

1. Main activities

The main activities are from procurement of raw materials to distribution and sales in the market. It consists of five activities: purchasing logistics, manufacturing, shipping logistics, marketing sales, and service. It includes activities related to the product itself.

Purchasing logistics

Raw material production and delivery: POIROT consists of enzymes, nucleic acids, Cas3-Cascade/crRNA complex, LFA strip, and plastic for the outer packaging. The enzymes are manufactured by NEB, and the Cas3-Cascade/crRNA complex is manufactured by Nippon Gene on an exclusive basis. The performance of these enzymes determines the performance of POIROT. Production at the industrial level requires a different technology than general culture, and external production is desirable for mass production and cost reduction.

Manufacturing

Product manufacturing and quality control: POIROT is manufactured at our company based on raw materials.Enzymes and nucleic acid are mixed and lyophilized. The POIROT technology itself is a combination of existing methods and is difficult to protect as intellectual property. However, by providing our production line, we can accumulate knowledge of enzyme preparation and lyophilization, as well as knowledge of specific sequences for various diseases, not just glaucoma. Therefore, POIROT will be produced in-house.

Shipping logistics

Shipping: The production base will be located in the Kanto region, where consumption is high. In Tokyo, real estate prices are not commensurate with the size of the land, so select a site outside of Tokyo with a good transportation environment. 4°C storage is required, and depending on the transportation distance, special refrigerated vehicles may need to be provided, which is not economically or environmentally desirable. Therefore, the product should be manufactured near the place of mass consumption and transported over relatively short distances.

Marketing, public relations: In Japan, products must not be advertised until as IVD products have undergone appropriate screening. Also, after approval, false hype must not be made. In order to gain the trust of users, it is important to clearly state that the product has been approved and the false positive and false negative rates. As a truly responsible product, it should never be advertised in a way that undermines trust.

Sales: It is expected to be used primarily in human health checkups and sold in pharmacies. In addition to the stable income expected from its use in human health checkups, it is possible to collect large scale data in a uniform manner. This would allow a large-scale investigation of miRNA concentrations in tear fluid, which could be useful for biomarker research.

Marketing

Public relations: In Japan, products must not be advertised until as IVD products have undergone appropriate screening. Also, after approval, false hype must not be made. In order to gain the trust of users, it is important to clearly state that the product has been approved and the false positive and false negative rates. As a truly responsible product, it should never be advertised in a way that undermines trust.

Sales: It is expected to be used primarily in human health checkups and sold in pharmacies. In addition to the stable income expected from its use in human health checkups, it is possible to collect large scale data in a uniform manner. This would allow a large-scale investigation of miRNA concentrations in tear fluid, which could be useful for biomarker research.

Services

Support: The epidemic of the new coronavirus has increased understanding of POCT devices by colorimetry. However, in order for people to judge the correct test results, the instruction manual displays how to read the results for each pattern of appearance of the detection lines. In addition, since the average consumer is not familiar with Schirmer test strips, it is necessary to inform consumers of the safe use of the test strips not only through text but also through video clips.

It also displays an introduction to an application that refers the user to a nearby eye doctor when a positive glaucoma test is suspected. Most importantly, POIROT is not a product that promotes undue anxiety about glaucoma. We also need to inform consumers that glaucoma can be slowed with proper treatment.

2. Support activities

Support activities are those operations that support corporate activities. They consist of personnel and labor affairs, technology development, corporate infrastructure, and procurement. It includes activities related to the periphery of the product.

Human Resources, Legal

POIROT is designed for rapid social implementation and is enzyme- and nucleic acid-based so that GMO regulations do not apply. However, there are several possible risks. One is the possibility of damaging the eye during the collection of lacrimal fluid. A legal section should be provided in case of such an eventuality. Another possible risk is contamination of the device. Since the Schirmer test strips are placed close to the eyeball, there is a possibility of bacterial infection from the mucous membrane or drug contamination from a malicious person. To address this risk, a quality control department should be established and quality inspections should be conducted on an irregular basis.

Technological development

Technology development is essential to POIROT; disease-specific nucleic acid biomarkers must be reported in order to improve the scalability of POIROT itself. This requires an exhaustive search, which POIROT is not good at, and we will work with research institutions that investigate biomarkers to expand the range of applications. We will also invest in the development of a lacrimal fluid collection method to improve the method of lacrimal fluid collection, which is the most risky in POIROT.

Enterprise Infrastructure

Corporate infrastructure often refers to IT infrastructure, which consists of hardware such as PCs and servers and software such as operating systems and applications. The security of the IT infrastructure must be improved to prevent experimental data from leaking outside the company, and for this purpose, a security system should be implemented and clear rules should be established within the company.

supply

The presence of stable funding allows the company to not only survive as a company, but also to develop new technologies and make investments; possible funding sources for POIROT include IPC, various grants, government startup support, and crowdfunding.

Growth Strategies

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Product “POIROT"

By recognising the assets so far, POIROT's strengths and weaknesses have been analysed. Based on the analysis, we will determine where POIROT is headed. This is because there is no strategy in haphazardly improving the product, and the analysis is meaningless. While it is important to be flexible and adaptable to the environment, it is also important to set a clear vision of what POIROT should be aiming for, which emerges from the analysis based on the users' opinions.

The first core part is disease detection, using amplification and quantification of disease-specific biomarker miRNAs. The goal has always been to not only conduct glaucoma testing but also to develop the capability to test for diabetic retinopathy ³³, as well as for challenging malignancies like pancreatic cancer and Alzheimer's disease, a leading cause of dementia ³⁴; for which biomarker miRNAs have been reported in tear fluid.

The next step is to design around the core: one is to improve the method of tear fluid collection and establish a method of collecting tear fluid using eye wash solution rather than Schirmer test strips. Although minimally invasive, safety should be the primary design priority, as the eye can be damaged. The other is to make the entire POIROT disposable as combustible waste due to growing environmental awareness. At present, there is no problem with the contents, and the use of biodegradable plastic for the exterior would solve this problem.

Finally, design services that are inclusive of users. Test results alone cannot cure a disease; they only provide one-way information. Therefore, a service to introduce and book hospitals near the user's place of residence should be developed as an accompanying service. Early detection is only worthwhile if there is early treatment, and it is our mission to arrange for the user to go to a hospital immediately after the test results are available.

Two possible forms of marketing are use in human medical check-ups and use at home. It can be used to check a wider range of health conditions, as it can address diseases that are difficult to find with current test items. It also encourages more people to go to hospital based on test results, so that treatment can be initiated in the early stages of disease. In Japan, annual medical check-ups are mandatory, which will provide a stable source of income for POIROT. As per the initial target, the device will also be marketed as an easy-to-use testing device that can be used at home.

Strategy

Leiden 2023 ³⁵ was helpful in our business planning. Their meticulous planning is a very good model for a start-up starting from synthetic biology.

The timeline for POIROT is presented below. For POIROT, a health-related product, to operate responsibly and successfully, detailed plans and targets should be established in advance.

For rapid social implementation, POIROT does not include GMOs in the device itself and aims to market it from 2029, five years later; although the time required for examination as an IVD product is uncertain, the business plan is focused on speed.

Standing in the market

Intellectual Property

Accumulating technology is essential in conducting business. By applying for a patent, which is a type of intellectual property, the invention is protected, allowing the right to file objections if a third party uses it without permission ³⁶.

POIROT is a novel technology that combines existing nucleic acid amplification methods with quantification techniques. However, since the foundation is based on existing methods, there were many uncertainties about whether a patent could be obtained. Therefore, we conducted Human Practicewith a lawyer knowledgeable in pharmaceuticals and medical devices, as well as with the TLO ³⁷that manages intellectual property at the University of Tokyo. When applying as an IVD (in vitro diagnostic) product, a new application is required for each disease, but this is different when applying for an invention. Since this is a novel method for amplifying and quantifying miRNA contained in tear fluid, there is a possibility it will be recognized as a patent. Data showing that miRNA can actually be amplified and quantified using tear fluid will be necessary for the application, so this will be demonstrated in future experiments. Additionally, a patent application must be filed within one year of the public disclosure, so considering the presentation at iGEM, the data must be collected and the application submitted by November 2025.

Stakeholder Mapping

The interests of users should be prioritized above all, but it is also crucial to consider the interests of various other stakeholders in order to sustain the business. The following stakeholder mapping is integrated with the Power-Interest Grid ³⁸, clarifying the position of each stakeholder in relation to POIROT.

The analysis was based on the Human Practiceconducted with both academia and non-academia individuals. The approach to the key stakeholders is described below:

• Civilization: The users of POIROT are general citizens, and their interests should be prioritized. To ensure they can use it with confidence, we will focus on improving testing accuracy and designing with safety and environmental considerations in mind. Additionally, we will provide accurate information to help users understand diseases correctly, especially targeting those in their 40s, a group where disease risk increases ³⁹. Although interest in diseases changes with age, we have chosen to make a clear distinction for this purpose.
• Medical Professionals: POIROT amplifies and quantifies disease-specific biomarker miRNAs for testing, so medical professionals will be directly involved. POIROT is expected to increase the number of patients visiting hospitals and the number starting treatment, which will raise interest but also potential skepticism among medical professionals. To demonstrate the superiority of POIROT over existing testing methods, we need to demonstrate POIROT's superiority over existing methods through clinical trials and show that a causal relationship exists between diseases and miRNAs. Gaining understanding from those who make the final diagnosis is crucial.
• Specialists of Tear Fluid: POIROT is based on tear fluid. Advances in tear fluid research will directly contribute to the evolution of POIROT. Investment should be made to advance both POIROT development and tear fluid research concurrently.
• Medical Checkup Companies: The companies providing medical checkups are the primary customers of POIROT and have the most significant interest. POIROT offers a substantial advantage by allowing easy testing of diseases that were previously difficult to detect using only tear fluid. These companies need to trust the reliability of POIROT's tests, and improving test reliability will likely lead to a positive consideration for its use, similar to medical professionals.
• Sustainable Organizations: Although they may not be interested in the test itself, these organizations are concerned about waste from disposable testing devices. It is important to clearly specify the materials used for the contents and packaging, their hazards, and disposal methods, and to ensure that the product is environmentally friendly. We will assert that POIROT is a sustainable product.

Rule-making

We discussed with a lawyer through Human Practice whether the current version of POIROT violates any Japanese laws or regulations. There were no aspects that required rule-making. This is because POCT devices using LFA, like pregnancy tests, have been widely used even before, and became familiar to the public during the COVID-19 pandemic. As a result, it is well-known both publicly and privately, and there is no need to create new rules.

In order to sell POIROT, it will need to be approved after applying as an IVD product. However, since it does not target any urgent diseases requiring expedited approval, there is no need for fast-tracked approval. Moreover, shortening the time for approval may lead to insufficient discussion or investigation, potentially causing problems. Therefore, changing the application and approval flow would not be a good approach.

Long-term Impacts

POIROT comes into our daily lives in terms of health. Whether or not individuals are aware of their conditions can have psychological impacts. As a truly responsible product, we must consider the long-term effects.

ELSI & RRI

POIROT does not make diagnoses but performs actions similar to declaring a condition. When patients receive test results at a hospital, the disclosure is often done with sensitivity, depending on the type of condition. However, POIROT outputs results without preamble. While the conditions identified are not immediately life-threatening, the mere knowledge of having a condition can pose psychological burdens on users.

In our Human Practicewith glaucoma patient organizations, we learned that patients in the early stages of glaucoma can sometimes experience depressive states upon being informed of their condition. Even if the condition is not fatal, the psychological impact on patients can be significant, and addressing this is crucial. As a responsible product, it is important to provide accessible and appropriate information about the conditions POIROT can detect, ensuring that users have access to accurate knowledge and support.

Other Risks

Another potential risk is dual use. Dual use considers the potential military application of the technology. We have addressed dual use through Human Practice and Education, and have discussed it with both experts and the general public. It has been determined that the risk of military repurposing of the components used in POIROT is minimal. However, future developments could introduce risks, and it is crucial to continuously manage and mitigate potential threats.

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