Oral cancer is a severe issue in Taiwan and still needs more public awareness. Therefore, our plans for human practice will not stop here. We will definitely continue on our journey to dedicate ourselves to improving the current situation of oral cancer in Taiwan. Our social media accounts will carry on to update the programs presently in progress. As for the podcast channel, we too are looking forward to accomplishing it. Last but not the least, we are constantly keeping in touch with the Sunshine Welfare Foundation, discussing the experience sharing of oral cancer patients. We foresee a near future that knowledge about oral cancer becomes common sense amongst the public, and miRNA.DOC evolves into a mature product. Perhaps by then, our human practice will be ready to step into the next page of promoting the healthcare brought by miRNA.DOC.
Introduction
We always hope that miRNA.DOC isn't just a collection of our lab results, but a project that can truly solve the existing problems in the society and can make a change in the world. During our visits to several stakeholders and professionals,
some provided us with different perspectives on this issue, while some gave us advice on their professional fields. Whether they inspired our design or helped us overcome problems we encountered, we actively incorporate those inputs in our project and it, finally, evolved into miRNA.DOC through the whole process.
We introduced and modified the design-build-test cycle similar to
UC San Diego 2018 iGEM team in order to shape our project. And our project was divided into three stages, determination, development, and deployment.
"Early detection is one of the keys regarding national oral cancer control."
Health Promotion Administration(HPA), Ministry of Health and Welfare
Health Promotion Administration is a governmental organization that was formed to be responsible for health promotion and non-communicable disease prevention work. It provides comprehensive health promotion services from womb to tomb and from families to communities. And the Cancer Prevention Division under it has been dedicating to cancer prevention by planning, executing, and supervising matters related to cancers.
Purpose:
To understand the issue from the governments' perspective in order to see how oral cancer has affected Taiwan.
Feedback:
According to the Division Director, cancer has been the No.1 leading cause of death in Taiwan since 1982, accounting for 28% of total death and 11% of National Health Insurance cost. And among all cancers, oral cancer has been a pressing issue due to its high incidence rate and mortality among young adults. Besides presenting statistics such as occurrence, mortality rate and related expenses in recent years, they also introduced policies and programs such as Taiwan Cancer Control Act and Taiwan's National Screening Program. They stressed how early detection can play an important role in lowering damages caused by oral cancer. By implementing government-funded biannual screening in high-risk groups since the 1990s, occurrence and mortality has gradually decreased.
Action:
We saw the importance of early detection and it aroused a lively discussion in our team about how we can improve or assist to optimize current detection methods.
"There exist problems in current detection method due to the detection bias."
Dr. Yu-Feng Huang, Dr. Yu-Chao Chang and Dr. Chun-Cheng Chen
All of the dental professionals are from Chung Shan Medical University, which is one of the hospitals in collaboration with HPA toward oral cancer issues.
Purpose:
To investigate the strengths and limitations of current detection method.
Feedback:
Currently, oral cancer is detected by visual examinations conducted by health care professionals. If OPMD(Oral Potentially Malignant Disorder) symptoms (ex. patches, lumps, or nodules) are found, those patients are referred for biopsies in medical centers as diagnosis. Though visual examinations are noted for their cost-effectiveness, those professors pointed out that there remains detection bias in the current method. For example, they shared that there is actually a low percentage of actual OSCC patients out of all biopsies. Also, those lesions might not be visible due to extrinsic factors sometimes, resulting in delayed diagnosis and the treatment would become more costly and difficult. Since visual examinations aren't quantitative, the results would rely heavily on personal experience, and may vary a lot depending on different people conducting them. In conclusion, a quantitative detection method is in urgent need to assist in current visual examinaitons.
Action:
This interview encouraged us to brainstorm how we can achieve quantitative detection through synthetic biology to increase accuracy and achieve early detection.
"We choose saliva as our biological sample for its proximity to the lesion sites of oral cancer."
Prof. Jau-Song, Yu
Prof. Yu is a professor in Molecular Medicine Research Center, Chang Gung University, who has dedicated in various biomarker research for decades. His team has also developed an immuno-based oral cancer detection kit.
Purpose:
The first important step in designing our device was to choose the biomarker and biological sample. Upon facing several biomarkers to choose from, we had a hard time narrowing down. Thus we reached out to their team for biological sample and biomarker selection advice.
Feedback:
He shared his experience of choosing biomarkers for his article "Saliva protein biomarkers to detect oral squamous cell carcinoma in a high-risk population in Taiwan". He divided the process into two stages, finding potential biomarkers and final selection with lab experiments. He finally narrowed down 4 protein biomarkers MMP1, KNG1, ANXA2, and HSPA5 from hundreds of potential biomarkers.
Action:
After taking Prof. Yu's advice and several research into consideration, we decided to choose salivary miRNAs as our biomarkers. We were also inspired by their biomarker selection method, deciding to list out various potential biomarkers and then narrow them down further.
"It's important to consider whether your biomarkers can distinguish between different oral cancer stages and their convenience for detection."
Prof. Cheng-Chia, Yu
Prof. Yu is a dental professor of Chung Shan Medical University expertising in non-coding RNA research, and has been the editor of several journals. .
Purpose:
After listing out several potential biomarkers, we reached out to Prof. Yu to assist us narrowing down our final biomarker selection.
Feedback:
He suggested us to choose upregulated miRNAs due to its detection convenience, and he also advised us on choosing biomarkers that can distinguish patients at different stages.
Action:
Combining all the advice and suggestions,
we finally picked our biomarkerssalivary miRNA 21,31 and 146 to not only distinguish healthy individuals and OSCC patients but also the different conditions between OPMD and OSCC. We believe that this could give healthcare professionals a more meticulous, quantitative, and objective method to diagnose OSCC patients.
Toehold switch
To measure the salivary miRNAs, we adopt a systematic ribocomputing devices, toehold switch. According to the research, a toehold switch is a secondary structure RNA with a hairpin loop that can regulate translation. Only with the specific RNA trigger can its structure be opened and start the translation of the following reporter.
"In order to minimize the leakage, a smaller loop is more ideal and I recommend you to use one of my more recent designs as a general guidance for the loop design."
Prof. Alexander Green
Alexander Green has pursued research in diverse areas ranging from synthetic biology to self-assembly to carbon nanomaterials with applications in flexible electronics, energy, and low-cost diagnostics. He also conducted postdoctoral research at the Wyss Institute at Harvard regarding toehold switches and was the lead author for several types of research on toehold switches..
Purpose:
When designing our initial set of toehold switches, we were struggling to understand some basic structures of the toehold switch such as the common linker and the loop structure.
Feedback:
We reached out to Dr. Alexander Green and he kindly offered to video chat with us to answer some of our inquiries. From the video session, he answered a lot of our concerns regarding designing a toehold switch such as designing our target binding site. He even explained to us the effect of the loop structure to us - the larger the loop the more leakage problem will occur. The loop contains the ribosomal binding site (RBS) in which ideally the ribosome would not bind to unless the toehold switch structure is unlocked by the target RNA. However, realistically, the ribosome would still be able to bind to the RBS randomly which causes the leaking of the protein. Green told us in order to minimize the leakage, a smaller loop is more ideal and he recommended us to use one of his more recent design as a general guideline for the loop design. Furthermore, he reassured us that changing the protein from GFP to our desired invertase would not have a huge impact on the functionally of the toehold switch as he has done it several times without any issues. Lastly, when we brought up that the use of the common linker he provided in his initial toehold switch paper would bind to our target binding site and create undesired secondary structures, he explained to us that the linker sequence can be randomized as long as it helps the toehold switch maintain its stability. He has introduced a common linker for everyone else to use in his initial paper for the sake of simplicity and easy replications of his steps. The linker sequence is just there to simply provide space to prevent domain interference between the loop and the protein sequence, so the sequence itself can be modified.
Action:
After the video session, we were able to drastically modify our initial sets of toehold switches based on his feedback and fix a lot of uncertainties to create our final versions.
Picture from: Cockrell School of Engineering, The University of Texas at Austin
To remove the equipment limitation on our detection tool, we did some paper research to find out the best reporter protein for our project.
Invertase, also called β-D-fructofuranosidas, is an important enzyme for the hydrolysis of sucrose to a mixture of glucose and fructose. It is commonly used as reporter proteins since its product, glucose, can be easily detected with a personal glucose meter (PGM),9 which has a high utilization rate in the public. According to the previous research, Thermotoga maritima Invertase (invertase from Thermotoga maritima) (TmINV) has been proven to have high activity and thermo-stability compared to the commonly used commercial yeast invertase.10
Thus, we chose it to be our reporter protein and decided to combine our design with the accessible quantitative device, glucometer.
"The experimental design would be more conclusive if negative selection is included."
Prof. Shun-Fa, Yang
Prof. Yang is the head of the department of research and development of Chung Shan Medical University. He has published over 500 researches and has conducted research on cancer detection and researches on miRNA.
Purpose:
During the time period where we started conducting our experiment, we have reached out to him for help regarding our experimental design as he has expertise in the field of miRNA and cancer detection.
Feedback:
He started by giving us feedback regarding our current research design and gave some positive feedback regarding the choice of biomarkers. Afterwards, he carefully went over our experimental design and gave us advice on how to make the research more conclusive. For instance, he recommended us to use a separate negative selection to test out the specificity of our toehold switches.
Action:
The negative control miRNA 191 was chosen as a response to his recommendation as it is also present in saliva and is quite stable across all individuals. Therefore, to ensure the specificity of our miRNA is not interfered with other present miRNAs in saliva.
"The RNA produced in the cell-free system can be easily degraded by environmental RNAse and therefore, would result in a low yield."
EPFL
EPFL 2019 iGEM team was the grand prize winner of 2019. They used toehold switches as the detection method to identify Flavescence Dorée , a disease that infects grapevine.
Purpose:
During the initial testing stage of testing our cell-free system with the application of toehold switch, we were encountering low production of signal levels from our reporter protein, green fluorescence protein (GFP). We tried to manipulate the different conditions and adjust the volumes of each material to attempt fixing the problem. We also tried to find experts in the field of toehold switch, but we could not find anyone to help us debug the persisting low production. While searching for help, we stumbled upon EPFL 2019 iGEM and their project which also utilizes a cell-free system with production of toehold switches. We sent an email and documented our current situation and tried to see if they could give us any guidance.
Feedback:
Luckily, they have also encountered the same problem previously and were able to fix the problem with the simple addition of an RNAse inhibitor. The RNA produced in the cell-free system can be easily degraded by environmental RNAse and therefore, would result in a low yield.
Action:
After adding RNAse inhibitor during our experiments, we were able to successfully express our toehold switches' reporter proteins.
"There is a big gap between research and commercialization that is hard to overcome by merely the strength of academia."
Prof. Jau-Song, Yu
Prof. Yu is a professor in Molecular Medicine Research Center, Chang Gung University, who has dedicated in various biomarker research for decades. His team has also developed an immuno-based oral cancer detection kit.
Purpose:
Initially, we were reaching out for advice on biomarker selection. However professor Yu also shared his experience of commercializing his research project and how his team collaborated with NARLabs Medical Device Alliance.
Feedback:
He shared that there are several trials to pass and analyses to conduct, which are hard to be completed merely by his research team. Thus he applied for assistance from NARLabs Medical Device Alliance, including services such as legal counseling and integration of media resources for exposure.
Action:
After conducting our experiments, we were inspired to discover more about entrepreneurship and
started planning our business plan. We also planned to visit the National Applied Research Laboratories (NARLabs) Taiwan Instrument Research Institute.
"We are expected to assist the products of newly-established teams to go from “innovation” to “creating value”, and lead the medical device industry to a prosperous future."
Taiwan Instrument Research Institute, National Applied Research Laboratories (TIRI, NARLabs )
TIRI is a government funded research institute pioneering in instrument related frontier researches foreseen by the nation. Their long term prospect is to advance national science competence and to improve the quality of lives for the public.
Purpose:
To understand how TIRI can assist research teams in commercialization.
Feedback:
During our visit to Taiwan Instrument Research Institute, they introduced their Medical Device Alliance and services they can provide and assist us. Its Medical Device Alliance was established in 2014, gathering the industrial, government, academic and research entities to accelerate the production of biomedical equipment in Taiwan. The Alliance is in line with the development characteristics of the Northern, Central and Southern Taiwan Science Parks and connects the three major medical technologies, including medical electronics, polymer medical materials and metallic medical materials. By professionally dividing responsibilities and integrating platforms across 20 members, this Alliance has come together to create a “one-stop service” to streamline Taiwan’s medical device industry.
Their services include
Trial Production of Prototype, Verification of Specs and Functions
Pre-clinicals and Clinical trials assistance
Listing Counseling and Financial Planning
They also shared different collaboration patterns between research teams and biotechnology companies such as technique transfer and industry-academia cooperation for us to choose from.
Action:
After consideration, we believed that industry-academia cooperation was a better choice for us due to the flexibility to keep modifying and improving the project. We started seeking biotechnology companies that are suitable for cooperation.
"We can assist in industry-academia cooperation and find resources for your future research."
Bionime consultant Kan-Jen, Tsai
Bionime is a leading glucose meter company dedicated to manufacturing Blood Glucose Systems for quality, accuracy and reliability, and its locations have spanned across the world. We have chosen to conduct our experiments with their glucose meter.
Purpose:
We presented our miRNA.DOC product launch and we would like to see the possibility of industry-academia cooperation.
Feedback:
We visited the consultant of the glucometer leading brands, Bionime, and demonstrated our product. Consultant Tsai gave us positive feedback on our creativity and product potential, he also said he could assist us in industry-academia cooperation and finding resources for our future research. Furthermore, he suggested components in our kit can be "recommended" instead of "sold" together in order to simplify the process regarding patents.
"Some high-risk individuals have problem opening their mouths for visual examinations"
Sunshine Social Welfare Foundation
Sunshine Social Welfare Foundation is a non-government organisation which aims to change the lives of people with facial disfigurement, as well as raising social awareness about the issue. In other words, the oral cancer patients they are assisting are actually those individuals who experience the most severe condition..
Purpose:
To gain insights from patients' and high-risk groups' perspectives.
Feedback:
High-Risk Groups of Oral Cancer include people who often consume alcohol, chew betel nut and smoke. Manager Chuang mentioned that the condition of Oral Submucous Fibrosis caused by betel nut-chewing, which is often seen in lots of betel nut chewer, holds them back from visual examinations, because they aren't able to open their mouths. However, with miRNA.DOC, it would be a lot more easier to collect their saliva for oral cancer screening.
Action:
After the visit, we realized that our detection method would be a blessing to lots of high-risk individuals. We were also touched by the social barriers behind this issue. Though there are problems we might not be able to solve in the short term, this interview reminded us to think about what kind of impact and engagement we students can make toward society.
"How do you overcome the complexity of salivary samples that differs from one another?"
Dental and ENT Clinics
Zu-shin dental clinic, San-Ho dental clinic and Tian-Ching ENT clinic are popular clinics near our school.
Purpose:
After the project design, we approached dental clinics and ENT clinics around CSMU in order to see whether there are considerations or feedback on our device.
Feedback:
Though problems such as biological sample complexity and little amount of biomarkers in it were still concerned, they said that installing our device in clinics would undoubtedly achieve earlier diagnosis, and clinics could be more engaged in oral cancer detection.
Action:
We were excited about the positive feedback from our potential users, and their concerns gave us some
future insights
on including biological sample pretreatments such as salivary purification and RNA amplification in our project.
