Results:

1. People's understanding of oncolytic viruses is low, even in the bio-related professional population, so we need to carry out more popularization and publicity of oncolytic viruses, so that the public can better understand the oncolytic viruses, and they thus can choose to use oncolytic viruses more confidently.

2. Although many people have expectations about the efficacy of oncolytic viruses, the vast majority of concerns still fall on safety, including the safety of use, the safety of storage, etc.

3. We hope to use influenza virus as the basis of oncolytic virus. Our current project aims to improve the targeting of virus to tumor, so as to improve its effectiveness and safety. However, such design cannot eliminate people's consideration of safety, so we need to strengthen the consideration of safety.

4. The production capacity of oncolytic viruses still needs to be considered, and the production yield, associated transportation costs, and storage capacity are still questioned, which may hinder the industrialization of our project

Expert Info:

Professor Xie Haiyan is mainly engaged in the research of new technologies and methods of biomedical diagnosis and treatment and has made a series of achievements in the fluorescence labeling of live viruses and the construction of biomimetic magnetic bodies and biomedical applications.

Record:

Mrs.Xie:What kind of protein was engaged in the process that influenza infected lung tissue? I wondered that whether your modification of the NA protein would affect the ability of the influenza virus to infect the lungs, thus making it unsuitable for targeting lung squamous cell carcinoma?
A:The philotropism of influenza virus to lung tissue was mainly due to the fact that influenza virus was prone to cross the lung mucosa, rather than due to a certain protein. Therefore, our modification of NA might not have a great impact on the lung philotropism of influenza virus. But in this regard, we would further investigate the literature or do experiments to verify it.
Mrs.Xie:Would the modification of NA affect the protein interaction?From my perspective,even though this approach made sense, there might also be undesirable effects.
A:It might indeed affect the protein-protein interaction as we increased the display ability of NA, and relatively weakened the display ability of HA, but the exact effect was still unclear, and further experimental verification was needed.

Mrs.Xie:I knew that in the article of professor Demin Zhou's team, they made use of the interaction between influenza virus and DC to activate the immune system to lysate tumor. But how did your influenza virus directly lysate tumor? Were there any side effects of this application?
A:We hoped to develop the influenza virus as a platform or carrier through this transformation. But as for the safety, we planed to involve some controlling systems so that we could get the flu virus replicate only in tumor cells and avoid possible side effects in normal cells. Besides，we had a question that as you professor have researched virus for many years,did you encounter any ethical issues related to the use of viruses before,or any problems about its safety and clinical approval?

Mrs.Xie:In fact, because of the outbreaking of COVID-19, our country was paying more and more attention to the safety of studying virus, relevant criterion or law would be stricter, and a four-level control principle was ready to form in our researching system.However, it was not about limiting our scientific applications, of course, but more stringent procedures were needed to ensure safety. For the oncolytic viruses,it received more and more attention in recent years, and more research would be done in the future. As long as we did it strictly to the regulations, there would be no trouble, and I believed that you team would make it!

Expert Info:

Professor Xiang’s main research interests are to establish and optimize viral infection and disease models (HBV and SARS-CoV-2), and to study virus-host interaction and pathogenesis.

Record:

Mr.Xiang: For the target TfR1 used in your experiment, do you investigate its expression in normal cells? As I wonder what is the minimum threshold for its functional effect, if the threshold is relatively low, will the oncolytic virus targeting platform you built have the "on-target, off-tumor" effect?
A: We have conducted a series of data research and analysis in the early stage, and found in many reports that there is a significant difference in the expression level of TfR1 in tumors and normal cells, but so far we do not have a very detailed data on the specific expression level. In addition, as for the question of the minimum functional threshold mentioned by you professor, we have not yet conducted research on it. Indeed, if this threshold is very low, the overall effectiveness may be greatly affected.

Mr.Xiang: I know there are many oncolytic viruses on the market nowadays, but why do you choose to use influenza virus as oncolytic virus? Can you tell me the reasons?
A: This is mainly because our professor Demin Zhou's research group has been committed to researching influenza virus and developing related vaccines for a long time, thus we have a relatively in-depth study on influenza virus. After we fish the NA protein of influenza virus capsid acting on the TfR1 target, we come up with the idea of using influenza virus to carry out targeted lysis of tumor cells. At the same time, in the specific lung squamous cell carcinoma model we select, we hypothesize that influenza virus could be administered by nasal drops or spray to achieve desired therapeutic effect.

Mr.Xiang: That's a great idea! But the question is, the strain you're using seemly fails to differ from the wild type, and how will you make it safe with this highly infectious viurs H1N1?
A: We have not yet studied this, but it is also a problem that we are most worried about. We can only say that every technology must have two sides, since we make use of its strong pathogenicity and strong targeting effect, we must consider its risk, but for this point thank you professor very much for your reminder!

Mr.Xiang: Have you considered the follow-up experiment arrangement? I mean, after this iGEM competition, will you continue?
A: We will make follow-up arrangements. To participate in this activity, it is enough to verify the targeting effect from the cellular level, but after coming back, we plan to conduct subsequent animal experiments to further evaluate and improve its targeting effect and safety. All in all, we are determined to make the influenza virus as a vector of oncolytic virus for targeted therapy.

(Student questions:)

Dingwei Lei : Professor, do you have any opinion on this kind of protein interaction modeling, or what aspects do you think can be started to conduct this modeling research?
Mr. Xiang: Based on your experiment, I think you can model from at least two aspects: firstly, the interaction between NA on the surface of wild-type influenza viruse and the target TfR1.It’s suggested that you consider their protein-protein interaction interface, configuration, the force of chemical bond formation and so on; secondly, you said that you have extended the stalk region of NA just now, so will this extension change the entire action mode? I think you can simulate the interaction between NA and TfR1 after the extension, so that the study will be more comprehensive and detailed.

(Summary)

Mr.Xiang: After listening to your project, I feel that what you are thinking and doing is very impressive! If you can really build a platform for targeting and treating tumors with influenza virus under the premise of fully considering the safety of influenza virus, then this is undoubtedly another major breakthrough for the development in the oncolytic virus field! I look forward to your good news and hope that you can communicate with me more frequently in the follow-up experiment!

Expert Info:

Professor Xie's research interests include building synthetic biology enabling technology platforms, using synthetic gene elements and gene circuits to study the design principles of transcriptional and post-transcriptional regulatory networks and the regulatory mechanisms of gene interaction networks, and building synthetic biological systems that monitor cell state changes in a non-destructive manner. He is committed to the research of biological gene function network and the key technology in the field of gene therapy application.

Record:

Ask:We have read your work Oncolytic adenovirus programmed by synthetic gene circuit for cancer immunotherapy published on Nature Communication that your team creatively established an artificial-controling adenovirus system to selectively activate the E1A protein expression in tumor, resulting in a more safe and effective oncolytic adenovirus therapy.And now,we were troubled by how to make sure that our oncovirus system(especially we used the influenza virus)is safe enough and convincing enough.Could you please offer us some suggestion?
Mr.Xie:The main consideration of viral safety was the regulation of viral replication so that it could only replicate effectively in cells that meeted the conditions, such as tumor cells; however, it failed to replicate in normal cells, thus enabling safe design of the virus.
From my perspective,such safety regulation could be started from the following directions, such as the conditional replication method used by your Professor Demin Zhou's research group, who made use of uncanonical amino acids and stop codons to insert virus genes to specifically regulate virus replication in tumors.

Answer:We have talked with Professor Zhou about the method of using stop codon to control virus replication, but as this could not achieve the purpose of tumor lysis functioned by the oncovirus, professor Zhou did not recommend us to use this method. However,we tried to come up with an idea that we hoped to improve its safety by truncating the NS protein in N1H1 virus, as reported by other group before, so as to make the virus lose its immunosuppressive effect in normal cell lines, but can only replicate in some malignant tumors,consequently more hypoinfectious and safer.

Mr.Xie:That’s right,and it is suggested that you team look up some literature to think about the feasibility of this approach. But as an influenza virus, it's airborne, and it's genome was the highly mutable RNA sequence, so if it were applied in patients, it could mutate into a new kind of virus beyond our expectation, and that risk needed to take into account.

Answer:It was exactly right! To tell the truth, we lacked some consideration in this regard before, so it was very urgent to solve the safety problem in our project.

Mr.Xie:Oh,an idea striked me that wheather you could also think about finding some specific inhibition or translation of initiation signals within the tumor to regulate viral replication. This method allowed the virus to be highly expressed in specific tumor cells and to replicate indolently in normal cells.Besides,I thought as you used the modified H1N1 to make oncolytic virus, it was necessary to evaluate whether the modified virus truly improved the destruction effect towards tumors. You needed to know how virulent it was compared to the wild-type virus.

Answer:Yes, we would add that point into our experiment schedule in the future.

(Summary)

Mr.Xie:Good job!It was hard to believe that you undergraduates were brave enough to challenge this tough project as it involved many difficult science problems.But I thought that all of you would get exercise of your ability to solve problems during countless try and fail.I heartfeltly hoped that one day you would successfully develop the influenza virus as a novel oncovirus!

Expert introduction:

Dr. Feng is the attending physician of the second Department of Thoracic Surgery, Peking University Cancer Hospital, and he had been to in Harvard as an exchange scholar, researching in the oncolytic virus field.

Record:

Dr. Feng: According to the project, I would like to ask what kind of administration you plan to use for the treatment of lung squamous cell carcinoma using influenza virus?
A: In the ideal case, we plan to use nasal drops for drug administration. This is because the research group led by Dr. Zhou has previously developed relevant replication-deficient influenza virus vaccines and proposed relevant drug administration strategies. We hope to use this non-invasive way for drug administration, so as to alleviate the pain of patients.
Dr. Feng: Yes, but this is a very ideal situation. In fact, the ability of oncolytic virus replication and infection is not so good as imagined. At that time, when I was doing relevant oncolytic virus projects abroad, we found that in fact, the ability of oncolytic virus to replicate and infect can go up to the third round, and in most cases, the replication of 1-2 rounds will be cleared by the body's immune system. At that time, we extracted the tissue and found that the concentration of oncolytic virus in the tissue was very low, and it could hardly play any role. Going back to your topic, if you are going to use the nasal drip strategy, have you ever thought that the oncolytic virus has to pass through the barriers to reach the tumor tissue, and there is no way to control the dose; In addition, there is no way to achieve targeted drug delivery, for example, if there is a tumor in the left lung but not in the right lung, then it is likely that only part of the virus will reach the left lung when it spreads, so is this a big challenge for the dose setting?
Answer: Indeed, after listening to your analysis, we found that the early preparation work is not very sufficient, at least we did not pay too much attention to this point, but stayed in a hypothetical stage, I think we will consider in the subsequent design of animal models and other verification.
Dr. Feng: In fact, there are quite a few ways of administering drugs in the lung, such as puncture, etc. You can check these methods, which will certainly be helpful for your design of animal models in the future.
After that our team members come up with some question...

Q: Dr. Feng, when we searched for the statistical data comparing the oncolytic virus drug therapy in China and the world in 2021, we found that the application of oncolytic virus in China is quite small; at the same time, we have also noticed that there are quite a large part of chemotherapy and radiotherapy in the treatment of tumor (for example, lung squamous cell carcinoma) in our country. How do you evaluate this?
Dr. Feng: Well, for any medical treatment, the first thing we have to consider is its safety, and this safety test takes time to verify. Since the emergence of the three clinical methods of chemotherapy, radiotherapy and surgery, they have undergone countless clinical tests and are confirmed to be safe and effective, so we will be more inclined to use these existing reliable methods. On the contrary, oncolytic virus is still a new field, and its research has made breakthroughs in recent years, but it is still not mature enough and has not experienced the test of time. Admittedly, foreign countries do do more in this branch, which includes two reasons, the first is that they can indeed develop new oncolytic virus drugs, the second is that many patients participate in the trial of these drugs, resulting in the frequency of use is indeed higher than that of our country. But in any case, I hope that in the future there will be a really good oncolytic virus drug as a beneficial tool to fight tumors.
Q: OK. Then, when we conducted a questionnaire survey, we found that some respondents were afraid of oncolytic virus, and they thought that using virus was not a good strategy. In your opinion, if an oncolytic virus drug is really developed in China in the future, will this fear of patients become a barrier to its application?
Dr. Feng: You're right, but not entirely. For a new thing that everyone does not understand, then naturally there will be fear; But you have to understand that the patient's emotions can be guided by the clinician, and that's what the clinician should do. When it comes to viruses, many people may have a negative first impression, but we can make them understand through positive guidance that we are giving them a seasonal flu virus that already has a coping strategy and has a very low death rate, and we can cite historical clinical success cases to convince them to accept our treatment. So I don't think this will be a step forward in the development of oncolytic viruses.
Q: So what challenges do you see in the development of oncolytic viruses, or using influenza viruses as oncolytic viruses?
A: At present, the research and development of oncolytic viruses are in contradiction. Obviously, for a new drug, without determining its safety and effectiveness, clinical studies mostly have to start from the third line and the fourth line, which means that a group of patients who come to trial may be unfortunate to progress to the advanced stage, they have experienced a lot of therapies, and their immune system is almost on the verge of collapse. At this time, it is difficult to obtain better results with oncolytic virus drugs; On the contrary, clinical trials of treatment from the first line, such as before surgery, are almost non-existent, but this also contradicts the system of developing oncolytic viruses, a contradiction between function and the premise of function.
For the use of influenza virus as oncolytic virus, I think it is both an opportunity and a challenge, predecessors have hardly used such viruses, their high infectivity and high pathogenicity make people worry about their safety, but I think after rational design and transformation, we can completely transform it into an oncolytic virus drug we want.

(Summary)

Dr. Feng: After listening to what you have said, I think the problems you are considering are very innovative and meet the clinical needs! Although I have never come into contact with influenza virus to make oncolytic virus before, I really hope to do this project with your team! I hope you can sort out and summarize what we talked about today and I will also consult more friends for advice. I am looking forward to our next communication!

Expert intro:

General manager and Technical Director of Shenzhen Yuanxing Gene Technology Co., LTD.. The company focuses on the research and development, production and outsourcing services of viral vectors (mainly adenovirus, poxvirus, lentivirus, etc.) gene therapy, gene vaccine drugs, cell therapy and other fields.

Record:

Q: Did you know about the administration of relevant oncolytic viruses before doing this project?
A: We have a certain investigation, at present, it is still mainly intratumoral injection, which requires high accessibility of tumors; But we also found some that were administered intravenously and intraperitoneally, and we think that's not a good way to do that, because it's likely that the immune system recognizes and clears out most of the effective oncolytic viruses, leading to a very high dose of the drug.
Q: Yes, you are right, so why did you choose the influenza virus as the oncolytic virus? We all know that the influenza virus has a natural infective activity in the lungs, but it is difficult to model this intrapulmonary tumor injection. What do you think about this, or do you have any better ways to administer it?
A: We chose the influenza virus because we had focused on this part of the previous research work in our laboratory and we had a deeper understanding of it; In addition, the confirmation that NA can interact with TfR1 strengthens the idea of using influenza virus as an oncolytic virus. As for the way of administration, we had previously thought about non-invasive administration such as nasal drops, but after talking to some teachers and clinicians one after another, we found that this was too ideal, and we are still looking for a better way of administration.
Q: So the idea is that you identify the virus strain first, and then you look at what tumors the flu virus can treat and you do some engineering, but actually there are very few influenza viruses used for treatment. As far as our company is concerned, the most oncolytic viruses we do are adenovirus, pox virus, herpes virus, etc., but in fact, for the development of oncolytic viruses, this is a very challenging work, so far the focus of the problem is still safety, effectiveness, only to solve the two discussions behind the marketization is meaningful. By choosing the flu virus, you've no doubt taken advantage of its natural affinity for the windpipe, and so it is. But I have a question, that is, some students said that by reducing or knocking down TfR1 can reduce the replication ability of influenza virus, but I have not seen the data; At the same time, in my understanding, influenza virus still has a broad spectrum of infection ability for epithelial cells, etc., so I would like to ask if you have a clear data on the relationship between TfR1 knockout and influenza virus replication ability, or how you can prove this selectivity? The above is the first question, the second is I want to talk about why the oncolytic virus is still in an early stage of development, which is mainly limited to targeting, including organ targeting, tissue targeting, and accurate targeting to cells. Only when it can be more perfectly positioned to cells in the end, can the oncolytic virus be used as a better drug. So I think you should also think about whether the oncolytic virus that you finally build can reach that last level?
For our company, I can share our modification of adenovirus. Because adenovirus must have the E1 gene to control its replication, we chose to add a switch in front of the E1 gene; This switch can only be turned on when a substance is highly expressed in tumor cells, which undoubtedly increases selectivity.
A: Your question is a very good one, that is, we did not give much consideration to the screening of strains. At the same time, in terms of the design of this switch, we also hope to consider the selective expression method you just mentioned in the future design, thank you very much!
Q: That's right, and according to the question I just asked, in fact, I find that you still lack a holistic thinking about this project. If you can complete this kind of switch design, it is undoubtedly the best, but if time is tight, I think you'd better write the whole thinking process clearly and completely, after all, in the definition of precision medicine, the lung is not a good target, which is still to be discussed. I also want you to explore the delivery system, because the results you see in vitro may be significantly different from the clinical results, which requires you to have a good foundation in disease immunology and clinical pharmacology.
A: Yes, what you said is very important! We have not considered this completeness and maturity before, and we will supplement and refine in this respect.
Q: In addition, I also hope that you can review the whole logic of the previous. In your PPT, you have seen a lot of comparison of differences in TfR1 expression, but in fact, since you are targeting lung cancer, whether the final focus should be on various cell lines of lung cancer, rather than a broad spectrum concept. At the same time, I think you also need to highlight the innovation and highlights in the competition, rather than needing others to help you sum up, I think this is also very helpful for you to participate in the competition. I have another question, that is, the influenza virus you chose is not very stable because it is an RNA virus, but when we market it, we hope that the whole thing is very stable in view of the control of product quality. I suspect that one of the reasons influenza has not been used as an oncolytic virus is that it is too mutational, so I hope you will think and design in that regard.

(Student question:)
Q: We also have a question about the market. We are still looking at our products from a laboratory perspective, but we are thinking if we look at it from a corporate perspective, a market perspective or a capital perspective, will this be different? We just want to ask what are the characteristics of a drug that can attract investment, that is, what other conditions are needed in the premise of ensuring safety and effectiveness?
A: You are asking the right person, the whole idea is an inverted triangle ladder way, the most important thing is to fill clinical needs, that is to say, what clinical practical problems can you solve, can provide new methods, or can reduce costs? You need to figure that out, and you need to spell out the real benefits for the patient. The second is to consider the survival rate, the most afraid of the problem is that the effect is very good but the patient still does not survive. However, this is also a very contradictory thing, because any new drug clinical approval conditions are to solve the high-risk population, that is, to climb up from the three or four lines, one of which is to look at safety, the second is to look at effectiveness, which means that the entire approval process is very strict, so we often see many drugs in the laboratory is very good, but in the clinical failure. Of course, I hope you will not blindly focus on marketing for the purpose, or you need to consider some diseases that are currently incurable, such as some rare diseases, and so on. I think if you can provide effective means for the treatment of these diseases, it will be better to expand the pattern!

(Summary)
General Manager Liu: After listening to your talk, I think the problems you are considering are very innovative and meet the clinical needs! Although our company has not come into contact with influenza virus to make oncolytic virus, we hope to follow you to do a good job in this project, and also hope that you can get a good result in the final competition! We hope that you can summarize the content of our conversation today and timely feedback to the experiment. We can set up a group chat and feel free to contact us if you have any questions. We are looking forward to our follow-up communication!

Expert intro:

General manager and Technical Director of Shenzhen Yuanxing Gene Technology Co., LTD.. The company focuses on the research and development, production and outsourcing services of viral vectors (mainly adenovirus, poxvirus, lentivirus, etc.) gene therapy, gene vaccine drugs, cell therapy and other fields.

Record:

Question:
1. What are the criteria for clinical approval? Effectiveness and safety depend on what?
Answer: The new drug IND application materials more reflect: molecular safety evaluation, whether to meet the quality compliance of scaled-up production and small-scale production, product formulation exploration and the stability of finished drugs

Answer: IND declaration does not need ethics committee, ethics committee need before the start of human clinical research, mainly used to give clinical experience to evaluate the advantages and disadvantages of experimental drugs and listed existing programs, clinical research design is in line with medical ethics, patients and family protection rights and interests in clinical research how to reflect through legal means in the specific medical situation judgment.

According to the Internet, the role of the ethics committee is to ensure the rights and interests of the experimental subjects, including the experimental scheme and the fairness of the subjects. What is the point of this?
Answer: Without the evaluation, protection and checks and balances of the medical ethics committee, it is easy for manufacturers to design radical clinical programs based solely on cell data or animal data, unilaterally pursuing a single clinical indicator (tumor size, inflammatory indicators, etc.), and failing to guarantee the overall survival benefits of patients.

What exactly is fair?
Answer: Prolong life and improve quality of life

Will the FDA review the test protocol?
Answer: Before initiating Phase 2, the FDA must approve the manufacturer's clinical study protocol and ethics committee report

6. What is the current mainstream experimental protocol for oncolytic virus phase II?
Background: T-vec uses Simon's two-stage design, which on the Internet is a conservative experimental protocol that is not very confident about its efficacy. Is that true of most experimental protocols today? Does this kind of clinical trial affect people's trust in drugs?
Answer: Phase 2 clinical studies are designed to ensure uniform phase 1 safety data, so most phase 2 protocols for breakthrough therapies are cautious.

7. Aiming at the preclinical experiment, T-vec was only tested on mice in this paper, and the safe dose was roughly estimated according to the volume of body fluids. At such times, do you need to work your way up, such as becoming a larger model organism? Two of the mice were mentioned to have developed fever symptoms, but the article said it was not caused by the oncolytic virus. How is this judged in clinical trials?

People in clinical trials are different, and the samples are not very large. How does this guarantee the completeness of the experiment?
Answer: A professional clinical CRO team, manufacturer CMO (chief medical officer) and clinical PI jointly designed the indications and evaluation indicators of enrolled patients.

What is our attitude towards Phase IV? Is the so-called efficacy is not good, safety is not good, so delisting is talking about clinical phase IV?
Answer: First of all, if the safety is not good (death of large animals, clinical phase 1), it will not be allowed to enter clinical phase 2; secondly, if the efficacy is not good (not better than the existing treatment plan, clinical phase 2-3), it will not be approved for market. Post-marketing clinical studies (phase 4-5) are more about expanding doses, expanding the original indication population, or "conditional marketing" requiring long-term follow-up of certain efficacy indicators. Since the FDA/EMA has strengthened the review of premarket clinical protocols and data of drugs, requiring the establishment of ethics committees, few products have been withdrawn from the market due to safety concerns in the past 20 years.

1. We have two possible delivery methods: nasal drops and intratumoral injection. Among them, nasal drops are relatively less safe, and intratumoral injection affects fewer places because it is directly ingested into the tumor. Clinical trials require different delivery methods Different clinical trials need to be established. Can the safety test of nasal drops be used as an upper alternative to intratumoral injection?

Answer: The effectiveness of drugs entering the body through the nasal cavity, blood concentration, peak time, metabolic rate in the body, therapeutic window, metabolites and other data in the two modes of administration, the impact on other cells of the respiratory tract, stability tests brought about by different dosage forms, and supporting equipment need to be studied by medical equipment.
Possible tests (cellular level) : Various possible contact cells of the respiratory tract

2. Is isolation necessary for the use of oncolytic virus? In particular, respiratory influenza viruses like ours are relatively more contagious, although there has been some improvement in safety. Answer: Biosafety data must be done and the match of the isolation device (dosing mode) must be initiated at the CMC stage prior to IND filing

How do nose drops in animal experiments map to clinical trial dosage? Is the respiratory tract of mice very different from that of humans?
Answer: Yes, the biggest challenge of oncolytic virus drugs is that animal data and human data are very different, clinical phase 1 data can not be reproduced in phase 2 clinical studies, so very experienced CMC experts need to optimize the virus, model exploration and a lot of work.

Can the safety test of nasal drops replace intraperitoneal injection?
Answer: No simple conclusions can be drawn, and controlled studies need to be designed

Expert Info:

Professor Xiang’s main research interests are to establish and optimize viral infection and disease models (HBV and SARS-CoV-2), and to study virus-host interaction and pathogenesis.