Expert Interview
Selection of Colorectal Tumor Features
When we discovered the main drawback of existing colorectal cancer treatments after reviewing the literature - the indiscriminate harm to normal cells (more details can be visited implementation ) we decided to identify the characteristics of colorectal tumors to specifically introduce parts and build engineered microbes for targeted therapy.
We originally wanted to achieve targeted therapy by means of immunization. It occurred to us that Dr. Yaofeng Zhao from the School of Life Sciences at Lanzhou University might be able to give us some help. His field of research is related to immunology and he has people in his lab working on immunotherapy for tumors. But during the communication with him, he mentioned the dangers and side effects of immunotherapy, such as bringing immune disorders to patients. We agreed that safety was a prerequisite for the project, so we discarded the idea of immunotherapy. Instead, Dr. Yaofeng Zhao inspired us that we could continue to dig deeper on the characteristics of tumor microenvironment, and we finally pinpointed three microenvironmental characteristics of colorectal cancer tumors after reviewing the papers, which are lactate, low pH and hypoxia.
It was this little conversation that set the tone for our project, and we are very grateful to Dr. Yaofeng Zhao for providing us with the ideas.
Introduction of Amplifying Genetic Switch
We previously just wanted to use three sensors without any additional parts. After reviewing the literature, we found that many challenges limit the clinical application of current biosensing systems: limited signal processing capability to synthesize multiple biomarker signals for accurate diagnosis and incompatibility of response time with diagnoses that require fast results. We were at a loss when we thought of Master Shuang Wang, who has extensive experience in this field, and we talked to her about our current situation. She suggested that we introduce amplified gene switch parts, saying that she had used them in her own experiments with good results, and we read the literature and found them very implementable.
Cell Culture
We have also encountered some difficulties in the experiment. The cells we passed always be contaminated, resulting in the morphology and number of cells not meeting the expected requirements. So we contacted Yalong Li, PhD., of the first hospital of Lanzhou University, hoping he could explain the cause of the contamination and demonstrate a standard cell passage method for us.
Dr. Yalong Li has rich experience in cell culture in toxicology related experiments of tumor drugs. After seeing the cells we failed to culture, he pointed out that our cells were likely to have received fungal contamination because white and light yellow dots were floating in the medium. The reason why the cells we cultivate will pollute a lot is that we do not do a completely sterile environment when we operate. He demonstrated a complete passage process for us, and after seeing our operation, he put forward the improper operation and precautions for us. After his guidance, the cells are no longer contaminated by our irregularities.
Capsule
Once we had the initial construction of the engineered microbe, we started to think about how to deliver it to the human body. Worldshaper-HZBIOX and NEU_CHINA first did background research on the current form of probiotic drug delivery, and after our discussion we decided on the oral form (more details can visit partnership). But after that many questions followed, did we end up with a dry powder? Will it pass through the stomach acid intact?
We chose to talk to Prof. Shouliang Dong, who works on peptide drugs. He suggested that we make the probiotics into a dry powder before putting it into the capsule, there are finished capsule shells for sale on the market but he found us a few references that we can use sodium alginate and chitosan to make our own capsule coating so that the ingredients are known to control its safety more effectively. More details about our capsule can visit hardware .
Public
Analysis of Questionnaire Results
At the beginning of our project design, we wanted to find out what every group in the community thought about colorectal cancer (CRC) and our project, so we distribute a questionnaire to the public through online and offline channels.
After a month-long questionnaire distribution, we eventually collected a total of 447 questionnaires from people from all walks of life in all ages. From the results of our questionnaire, we know that nearly 70% of the answers are composed of people aged 18 to 30. Among them, 95% of respondents had at least received high school education, and their work was very different.
In the following questions, we explored the public's understanding of colorectal cancer and its treatment. It was found that nearly 90% of the public has a certain understanding of colorectal cancer, but about 40% of them are completely ignorant of its treatment. Then we ask what do you think is the most important concern in the treatment of colorectal cancer this question. This is to make it easier for us to improve our protocols and make our treatment more acceptable. Then we found that efficacy and safety were the most common concerns, which was consistent with what our team had always thought.
After learning about this information, we briefly introduced our project to the investigators in the form of cartoons—using gene-editing technology develop a targeted probiotics to treat colorectal cancer. After a brief introduction to our project, we asked the investigators further. We found that 66% of respondents expressed support for our technology, mainly because they believed in our safety and efficacy; But at the same time, a third of the people are on the sidelines of our technology for economic reasons and lack of understanding of our methods.
In addition, we asked the fillers to rate their attitudes towards GM foods according to their own, with 1 being unsupportive, while 5 being supportive and 3 being neutral. The result was 3.19 points, indicating that most people have a more conservative attitude towards GM food, thus suggesting that the public may have the same attitude towards GM probiotics, as both are meant to be eaten in the stomach.
At the end of the questionnaire, we set up two questions about synthetic biology to understand the public's perception of synthetic biology. The first one is “Have you followed events such as the Resurrection of Mammoth, the Human Embryo Gene Editing Project, the gene-edited pig heart that can be transferred to the human body, the synthetic starch, and the artificial meat?” Indicating whether the general public is aware of synthetic biology by asking them if they follow these genetically modified hot topics. The results show that about 90% of the respondents have some understanding of synthetic biology, but only a small number of people have a deeper understanding of it. The latter conclusion we reached by asking the second question “Do you think there is a difference between transgenic technology and hybridization technology?”
Promotion to Our Project
We found that the most popular reason for not supporting our engineered probiotic was for economic reasons, so we chose the most accessible and harmless E. coli as our chassis to reduce the cost of future mass production. We originally selected only one therapeutic protein, CCL21, as our therapeutic part, but the public is most concerned about the efficacy, so we added two other therapeutic protein components by reviewing the literature, namely HlyE and CDD-iRGD, more detail can visit project description. Although we have only performed in vitro experiments, we can compare the therapeutic effects of the three proteins in subsequent in vitro experiments to select the best one or combine them.
Finally, we also found that the public has only a general understanding of synthetic biology, so we have launched educational activities for different age groups to reach out to as many people as we can, more details can visit education.
Government
On June 16, 2022, our team went to visit the Gansu Provincial Medical Products Administration, a government agency directly under the state. Because the results of our experiments are expected to enter the market in the form of probiotic drugs.
Before going to the visit, as it was the first time for us to contact with the drug supervision and management department, we made some preparations in advance to make the visit more smooth. We mainly wanted to know the process of launching probiotic drugs and cancer treatment drugs in China.
We were warmly received by the Gansu Provincial Medical Products Administration and were able to solve a lot of confusion according to the relevant laws and regulations. Through the visit, we learned that, as far as Gansu Province is concerned, it has been about ten years since a new drug was successfully registered, and probiotic drugs, as non-traditional drugs, will be only a few more years since the last registration of the same type of drugs. Regarding the criteria and requirements for medical products listing, the receptionist introduced us the details in the chapter "Drug Management" of the < Regulations for Implementation of Drug Administration Law of the People's Republic of China >.
At the same time, each medicine must go through a strict approval process, requiring non-clinical trials and clinical trials in the case of clinical drugs, which must be conducted in state-certified drug non-clinical trial institutions, drug clinical trial institutions, and comply with drug non-clinical research management norms, the test data obtained will only be recognized. (<Drug Administration Law> Article 17 engaged in drug development activities, should comply with the drug non-clinical research quality management norms, drug clinical trial quality management norms to ensure that the entire process of drug development continues to meet the statutory requirements.)
When we asked whether there is a marketing incentive policy for probiotic drugs or anti-cancer drugs, they specifically pointed out that there is a priority approval process for anti-tumor drugs according to Section 3 of the <Drug Registration and Administration Measures>, and that the country has some policies to accelerate the marketing of currently untreatable diseases.
Before our visit, we thought that market research and positioning of drug prices were also part of the work of the Medical Products Administration. After inquiry, we learned that market research is the act of R&D personnel, if data is available, it will be reported directly to the State Medical Products Administration by R&D companies, while positioning of drug prices is the work of the Market Supervision Administration of the National Development and Reform Commission. We also learned that the availability of sufficient funds is an important influencing factor on whether new drugs can be developed. The lack of funds of enterprises are the important reasons why no new drugs have been submitted for approval to market in Gansu Province for about ten years, but it is worthwhile to be happy that some enterprises have submitted for approval in the past two years and are expected to get perhaps 1-2 approvals this year or next year.
A surprising tidbit is that during our visit, we discussed about the classification of drugs, which can be classified into chemical drugs, biological products, and traditional Chinese medicine according to the registration category (Article 4 of the <Drug Registration Administration>), and the other side raised an interesting question about the research project we did - is the edited probiotic considered biological products or medical devices? We explored this question after the visit. But unfortunately we could not get a very definite answer even after reviewing a lot of information, so we sent an email to the most authoritative institution in China - National Medical Products Administration - for help.
After waiting patiently, we finally received a reply from National Medical Products Administration. The staff there called us and explained to us that, based on the materials we provided, they could tell that the probiotics we had edited were not therapeutic through physical action and did not meet the definition of a medical device in China, and therefore should be a biological product rather than a medical device. Our doubts were finally resolved. It also pointed the way for us to put the probiotic into the market afterwards, such as what tests should be conducted and what documents should be filled out.
Patient
As a team working on a treatment-related project, we agreed at the beginning of the project on interviewing the patients concerned - a vital and essential part of the process.
We tried to contact the First Hospital of Lanzhou University early in the season to communicate with them about various aspects of interviewing patients. With the help of Yiqing Wang, we were able to find out about the patients from the physician we contacted. Through the physician, we were able to get a more realistic view of the patients at the hospital, especially about how well the patients knew about their illness.
According to the doctor, some patients do not know that they have colorectal cancer because of their families, and they think they just have polyps or enteritis. In addition to the level of knowledge about the disease, he also gave us more details about the patient's education level and financial situation. It is fair to say that these realities were very different from what we had assumed. This meant that the interview script we had prepared earlier was not entirely appropriate for the patient we were going to interview and we needed to make a lot of changes. On the advice of the physician, we completed a new interview script, after learning more about the patient's situation, we wrote two interviews with people who knew about their condition and those who did not. It was a lot of work for us to make these changes, but we felt it was worthwhile and rewarding: we interviewed patients to understand more about the audience for our treatment program and to draw out their concerns about treatment options from their answers, so that we could improve our project in a more targeted way. At the same time, we want to treat each patient with respect and, as far as possible, not to disrupt the rhythm of their treatment and their daily lives. After a long wait, the interview protocols and scripts we submitted were finally approved by the Ethics Committee of the First Hospital of Lanzhou University. Here is the transcript of our interview.
After much coordination, we finally got to actually come to the patients at the end of September. We were divided into two groups and interviewed four and five patients respectively. It is important to note here that the ethics committee of the First Hospital of Lanzhou University agreed to our interviews and asked us to respect the privacy of the patients. Therefore, we did not keep records of the interviews that might reveal the privacy of the patients, such as photos or recordings.
We were all very nervous at first, fearing that we might say the wrong thing, but these nerves dissipated when we stepped into the patients' wards and were greeted warmly by these upbeat patients. To our surprise, almost every patient was very willing to talk to us, and we actually talked about a lot more than we had prepared in our interview scripts. At the same time, we were pleasantly surprised by the number of questions we encountered, such as the exact difference between E. coli and yeast, what are the risks of clinical trials, etc. We answered their questions as thoroughly and exhaustively as possible. As mentioned above, we did not leave photos or recordings in order to protect the privacy of the patients, so we could only record our interviews in writing on the spot. Basic information about each patient, such as age and occupation, was obtained from the treating physician prior to interviewing them, but this information was not kept on any records as a way of protecting the patients' privacy. We spent at least half an hour in each patient's room talking to them.
When we returned to the school to compile the interview notes, we found that all the patients coincidentally mentioned one topic - the cost of treatment. Due to Chinese etiquette, we did not ask each other directly about the financial aspect, but patients believed that the cost of their illness is what makes it almost unbearable for them or that it is the cause of the great emotional stress they bear after their illness. We also found that they had some prejudice against E. coli, which they perceived as an unclean strain. Compared to yeast, E. coli was very unpopular with the patients, and the words they used to describe E. coli were almost unanimous, such as "disgusting", "dirty" and "unhygienic", which we speculate that this may be related to their health education, as they generally consider E. coli to be a harmful strain of bacteria that causes intestinal disease, rather than a generic term for a class of strains. This greatly inspired us to follow up with an active promotion of education to eliminate the negative image of E. coli in the public mind. We would also like to make a suggestion for future teams: if the drug being made is to be taken orally, consider yeast as a chassis organism. After all, our ultimate aim is to improve our project into something that is widely supported by patients and can be promoted on a large scale in society. We have also partnered this time with NEU_CHINA, which uses yeast as a chassis organism to treat colorectal cancer.
What’s more, the patients all tended to be conservative in terms of their level of willingness to use our treatment in clinical trials, scoring around 5 (0-10), with the highest score being 7 and the lowest 1, which was in line with our previous expectations. Their concerns were mainly about safety. So we introduced the killing switch and capsule in a follow-up experimental improvement. The feedback we received from the patients through the interviews allowed us to continuously modify our project and work towards patient satisfaction.
When we asked them later in the interviews for their suggestions for our treatment program, the two points they stressed were almost unanimous: to keep the price of treatment within the patient's reach, and to minimize the pain caused to the patient. This provided important information for us to continue to develop and improve this treatment program at a later stage. This interview also allowed our team members to understand more about the mental and psychological conditions of the patients and to really get out of the lab so that our project can be more relevant to the needs of society and so that this project can have the opportunity to make a real difference to society.
Team communication and cooperation
April 28th : Meeting with NEU_CHINA
Since our two teams have been working together since last year and the team members know each other, and it so happens that their project this year is also related to colorectal cancer treatment, so we have established a close relationship since April. In this meeting, we shared our initial opinions of the project, we were inspired by their design --- let their RTAC have the ability to capture RSPOs by adhesion. We didn’t have an adhesion part at that time. Therefore, after discussion and literature finding we then decided to add the adhesion part: HIpA.
May 1st : iGEM Meeting Up
The iGEM Meeting Up was an exchange meeting organized by ZJU-China, CPU_CHINA, ZJUintl-China, JUT-China, HiZJU-China these four teams. Due to the impact of the epidemic, we were forced to choose to carry out online in the form of Tencent Conference. In addition to these four host teams, 20 teams from this year's iGEM competition, including our LZU-CHINA, participated in the event.
This was the main brochure of our meeting. It contains the specific process of the meeting and the team profile.
This is the team brochure, which records the logo of each team and the contact information of the person in charge of the meeting to facilitate communication.
The meeting began promptly at 4 p.m. on May 1st. After introducing the guests and the participating teams, each team will report on their projects in the order of the participation manual. Our team was introduced in the fifth stage, and the person in charge of our team, Zheng Huang, made a wonderful report at this event.
At this event, we got a lot of valuable advice. We just sorted out the general experimental ideas in May, for the HlpA adhesion experiment we originally designed to observe only under the microscope, the member from ONCE-CHINA suggested that quantitative experiments should be added, so we subsequently added plate counting. This meeting not only allowed us to learn about the projects of many other teams, but also to refine our own experiments, which was very meaningful.
June 25th: iGEM North-South Forum
Thanks to the organizers Sustech_EMB have been working hard to promote the first iGEM North-South Forum. On June 25th, the forum was successfully held, and 10 teams, including the organizers, participated in the conference. This forum is so named because the organizers invited teams from the south and north of China.
This is the general arrangement of the participation of this forum.
This is a project presentation for all ten teams participating in the conference.
This exchange inspired the integrated human practice, and in the meeting we gradually realized that the government and other regulatory departments also play a very important role in the development of probiotic drugs. Therefore, we added an interview with the Gansu Provincial Drug Administration in the follow-up human practice and achieved good results.
July 16th: China iGEM Online Meetup
The conference was co-organized by four teams: GXU-China, JLU-CHINA, Jiangnan_China and NPU_CHINA. At this event, each participating team has enough time to introduce their projects and communicate with interested participants, mainly through online meetings. At this event, our project also received a lot of attention and support. Through this event, we also found that our project has insufficient attention and concern for the vulnerable groups in society, so we have carried out a series of targeted activities to improve our education.
This is the online manual of the event, which introduces the project content of the 27 participating teams in more detail.
July 23th Northwestern China iGEM Meetup
As we are located in the deep inland northwest of China, it is difficult to find collaborations in synthetic biology. We are honored to participate in this Northwestern China iGEM Meetup, so that geography is no longer a limitation for the meeting of ideas. This meeting was hosted by NWU-CHINA-A, we are joined by NWU-CHINA-A, NWU-CHINA-B, NPU-CHINA, XJTU-CHINA, SZPT-CHINA, and SZ-SHD.
Before the conference, we prepared presentations and rehearsed our speeches in advance to let other participants know about our project in the shortest and most understandable way. We actively communicated with other participants and tried to invite as many teams as possible to our conference to expand the impact of our project.
After a brief exchange of pleasantries, we started our activities. Each group's session consisted of two parts: a presentation session and a question-and-answer session. During the presentation we learned about many well-conceived experiments and hardware, and during the question-and-answer session we obtained new inspiration for our modeling. This meeting promoted the collaboration in model construction between us and NWU-CHINA-A.
This event was very meaningful for us. We met many teams working on gut microbiology through this event. This allowed us to have a deeper exchange of ideas, such as biological parts, education, and partnership. This meeting laid the groundwork for further improvements to our project.
