Human Practices
"Human Practices is the study of how your work affects the world, and how the world affects your work."
Peter Carr, Director of Judging
Human Practices was one of the most important aspects in our project and since the beginning was the factor that shaped our project the most as PHANGEL aims to find application in humans. Read all our considerations below.
In the very beginning of the iGEM season, when we were just beginning to find and develop a project idea, our iGEM instructors gave us several presentations about topics relevant in synthetic biology. One of them, held by Prof. Michael Sauer, was about Human Practices and the responsibility of scientists towards the world. When working with GMOs, three key values have to be considered (but on a more philosophical level these might be applicable for most actions):
Safety
Responsibility
Benefit for the world
Modifying an organism just for the sake of the modification is generally not desirable. Naturally, but even more encouraged by this lecture, we were trying to find a project that not only pursued scientific knowledge but served a humane purpose.
As already explained in our project description, we believe the antibiotic resistance crisis is an urgent problem affecting the whole world and it cannot be researched too much.
However, it is also a big and very complex problem that goes beyond the scope of one iGEM project. Discussing this problem brought us onto the path of phage therapy. As described in the project description, bacteriophages are viruses that target bacterial cells only and their clinical application in western countries is on the rise. But often when we read something about phages in therapy it was mentioned how they lyse the bacterial cell and the endotoxin LPS is released.1 As already explained in the project description, LPS can trigger severe immune reactions ranging up to a septic shock. Literature suggest that a genetically engineered bacteriophage might be able to overcome this risk.1
With this in mind our project started to take shape. What if we could develop a therapy for sepsis patients that was independent of antibiotics? Could we genetically engineer a bacteriophage that not only kills the infection-causing pathogen but also prevents the harmful endotoxins from being released into the system? We wanted to try and developed the concept for PHANGEL that comprised all three values that we had determined in the beginning.
Safety
Beside the due safety measures the scientists have to take in any lab and the additional precautions caused by SARS-CoV-2, which you can find out all about on our Safety page, we also made our project design safe:
The integration of a safety mechanism in our project design was an absolute necessity for us since we aim for our GMO to be applied in the human body in the future. With the help of an appropriate safety mechanism we will not only ensure control over the given dose in the treatment of patients but also increase acceptance within the population.
Our solution was to disable the phages from replicating autonomously. The gene coding for the major capsid protein 10 in the phage genome which is essential for phage assembly is replaced by our transgenes. Without this gene, the phages cannot replicate outside a special bacterial phage-production strain that carries the major capsid gene on a plasmid. This mechanism is highly improbable to fail because our transgene for gelsolin is unlikely to mutate into the major capsid protein or an equivalent protein within one generation. Beyond that, this safety control can easily be transferred to other kinds of phages and makes a potential admission as a therapy more likely.
Responsibility
As GMOs are a topic that polarizes society and is afflicted with a lot of prejudice we needed to be careful when addressing it. At the same time we, as members of the scientific community, felt the need to raise awareness to the seemingly endless possibilities of synthetic biology and promote our project. In the tab Science Communication you can read how we tackled this. During our talk “Get Cozy with PHANGEL” we discussed GMOs in general and GMOs specifically as a therapeutic option with the audience. The responses were, as expected, quite mixed and we tried to answer the audience’s questions as good as we could. GMOs offer a bright future in many fields in science but science should not lose acceptance of the people whom it is trying to bring benefit to. We see it as one responsibility of scientists to encourage an open conversation.
As a team we also felt a responsibility towards each other. We tried to make sure that the communication within our team was good, that everyone was informed about the project and felt comfortable to speak up when something was unclear to promote an open policy and through that avoid mistakes. This was a lot harder this year as the lockdown prevented us from real-life meetings. We agreed on regular online group meetings and a weekly “Journal Club” in which the theoretical background of our phage therapy project should be split into different parts. Each group member was assigned one topic, that they had to research in literature and present the essential information e.g. what is currently known about it, which techniques are used and in what direction the research will go in future. The fundamental ideas behind this strategy was to bring us to the same level of knowledge of theoretical background (we all study different subjects) and to strengthen the group spirit during these difficult times where just online meetings were possible.
Benefit for the world
As described in our page about proposed implementation and the project description, our project aims to help patients suffering from a sepsis caused by bacterial infections. Since we are using a bacteriophage, the project is especially relevant for infections caused by multi-drug resistant bacteria for which no treatment is effective anymore.
Because it is easy to create a bubble around yourself and your problem and lose the overview over the real life value and applicability of your project, we reached out to different kinds of experts in the field of phage therapy, LPS and septic shocks. The feedback we received offered a lot of insight to us and shaped our project further. In the following you can read about these talks and how their integration shaped our project.
Disclaimer: All interviewees have been informed about the content of the interview, the purpose of the interview and what their answers would be used for. All interviews summarized here have been conducted with prior informed consent. Unfortunately, the medicinal microbiologist we interviewed went on a holiday right when we wanted to ask him to approve the final version of our summary of the interview, hence we will not mention him namely. However, as he gave us valuable insights into his field that had an impact on our project, we find it crucial to share it in the context of human practices.
Biomin
On July 8 we presented our idea for the first time to our sponsor Biomin. Biomin is a sponsor of the iGEM team in Vienna and presenting to them is an annual event and highlight in the iGEM season in Vienna. This year, however, was the first year we were not able to present in person. They are always interested in the iGEM projects, but this year they could especially relate:
Biomin is an Austrian animal nutrition and health company, specialized in the production of feed additives for poultry, pigs, ruminants and aquaculture. As LPS intoxication is also an issue in livestock, the staff of biomin already understood a lot about the background of our project. Animals are naturally in contact with higher LPS concentrations and are generally less reactive to it. Their gastrointestinal tract is a good barrier and a healthy epithelium prevents LPS from entering the bloodstream. Several factors such as nutrition and stress can impair this barrier and increase the LPS transfer over the epithelium. Also contaminated feed or concentrated feed can cause an increased exposure to LPS.2 Another risk factor for animals but also humans is the higher amount of LPS in air and dust in animal stables and its entrance through the respiratory system.
Biomin has specific products that tackle these issues: Some of their feeds contain additives that adsorb endotoxins and thus prevent their entrance into the bloodstream.3
Thank you, Biomin, for welcoming us once again!
Food for Thought from PhagoMed!
On August 10, we had a video call with Alexander Belcredi, Co-founder and Co-CEO of PhagoMed. PhagoMed is a Vienna-based biopharmaceutical company developing endolysins and phages as therapeutics. The company focuses on creating new treatment pathways for debilitating diseases such as recurrent bacterial vaginosis or implant-associated infections.
We presented our project idea and how we imagined its implementation and received some valuable feedback on what aspects we still needed to consider to ensure our project is applicable in the “real world”.
For once, they made us aware of the fact that one advantage of phage therapy is the potentially easier dose regimen. As phages only replicate in their specific hosts, therapeutic phages will in theory replicate as long as the infection-causing bacteria are present. Once the host pathogen is gone, the phages will no longer be able to proliferate and will over time be degraded and cleared from the human body. Our integrated safety mechanism that impairs the assembly of a second generation of phages after application, undoes this advantage. Since only one generation of phages is available to fight the infection, the dosing regimen becomes more complicated and sufficient bioavailability at the site of infection becomes a priority.
This was an important objection to our idea and it was followed by intense research and reconsideration of our safety mechanism. In the end, we decided to keep the mechanism as it was because beside this obvious disadvantage, it also possesses some advantages:
It is hard to disable this safety measure through mutations of the phage genome because we think it is unlikely that our transgene for gelsolin will mutate into major capsid protein within one generation and thus enable the phage to self-replicate.
It is a safety measure that can easily be transferred to other kinds of phages.
It facilitates a potential admission as a therapy because it cannot self-replicate.
It can be applied in a passive treatment regime which is not uncommon for phages.
Another topic that arose during our talk was the legal situation for phage therapy, especially genetically modified phages. Natural bacteriophages are classified as pharmaceutical biologics by the EMA and FDA. In the European Union phage therapy can be received under the Compassionate Use program if all other treatment has failed and in Belgium a decision was made in 2016 that allowed pharmacists to process natural phages.
The legal framework for GMOs in Europe, however, is rather restrictive and the potential release of GMOs might complicate the admission for clinical trials. We perceive this as an issue that will be dissolved over time and with the occurrence of even more research as the first steps towards phage therapy are already made: In 2018 a 15-year-old boy was successfully treated with genetically engineered bacteriophages against chronic infection of drug-resistant Mycobacteria.4
We hope that our specific safety mechanism will enable a potential admission of our GMO phage. We also discussed this later again with Dr. vet. med Sandra Wienhold, a phage specialist at Charité in Berlin (see below).
We took the feedback session very seriously and used the suggestions to further improve our approach.
Thank you very much to Alexander Belcredi and PhagoMed for taking the time and challenging our approach!
Our Interview with a Microbiologist
In September we had an interesting interview with a medicinal microbiologist. Unfortunately, he went on a holiday right when we wanted to ask him to approve the final version of our summary of the interview, hence we will not mention him namely. However, as he gave us valuable insights into his field that had an impact on our project, we find it crucial to share it in the context of human practices. Read the summary of the interview below.
Due to the constant increase of antibiotic resistances in microbes, more and more medical professionals are opting for a phage therapy. But from “bench to bed” it’s a long way and many questions need to be solved to generate a safe and high effective cure.
To solve questions regarding our project, we got in touch with a medicinal microbiologist. We talked about the emergence, current medical methods and prospective solutions in phage therapy. We also received valuable contacts for further interviews.
Phage therapy was developed in the former Soviet Union and since then further improved in states like Georgia (Europe). In Tiflis, the capital of Georgia, is the world’s most specialized hospital for phage therapy, the Georgi-Eliava-Institut for bacteriophages, microbiology and virology. In the Western part of the world phage therapy was neglected for decades, and only gained new popularity with the rise of antibiotic resistant microbes.
In phage therapy there are two important steps: first optimizing the therapy and second, ensuring that the patient suffers no septic shock.
To optimize the phage therapy, a cocktail of different phages, specially tailored for the patient, is used which targets just a single microbial species. This cocktail is necessary to ensure that even mutants of the targeted bacteria species, which developed a natural resistance against a certain phage species are infected to avoid its escape and further rampage through the body.
Avoiding a septic shock is also a crucial point in phage therapy because in the worst case it can end deadly for the patient. Septic shock is a strong innate immune reaction, triggered by LPS which is a part of the microbial outer membrane before its lysis by the phages. To prevent the septic shock, patients are currently treated with liquid infusions, usually of noradrenalin or epinephrine, to ensure physiological stability during their therapy.
Binding the LPS with a protein encoded by the phage to avoid the immune reaction could improve a phage therapy. The patient would not need further treatment with infusions which would in turn reduce the costs. However, it will be a long and difficult way, until such a genetically modified phage gets approved for medical usage.
Thank you very much for your insights, advice and contacts which helped us a lot!
Talk with Dr. Wienhold
Our time in the lab was already over when we had the honor to talk with Dr. med. vet. Sandra Wienhold about our project. Thus, we focused on the next steps in the pursuit of this project in this interview. Dr. Wienhold is a scientist at the Division of Pulmonary Inflammation at Charité in Berlin, Germany and responsible for the preclinical testing of phages. She is, therefore, an absolute expert for our specific questions.
Before the interview, we listed the questions that we wanted to hear her opinion on, especially to learn about her clinical experience, and sent it to her. These questions served us as a guideline for the interview:
Who were the potential users of our approach? We initially thought about older and/or immunosuppressed patients that would profit most from septic shock prevention.
How could the phages be applied?
Can the phages survive a passage through the stomach? We read that they are most stable at pH 6-8 and lose their activity at pH<3.6
Which safety measures need to be taken when handling or applying genetically modified phages?
How high is the risk for the bacteria to develop resistance against phages?
Is LPS intoxication an actual problem in the application of antibiotics and phages?
Is the application of LPS binding proteins common in the treatment of sepsis?
Is LPS really rendered harmless by LPS binding proteins? What would happen to this complex in the blood? Would it be metabolized?
Do you see an advantage of our approach compared to current therapies?
Beside answering these questions, Dr. Wienhold gave us a lot of input regarding the design of a potential clinical study in the future:
First of all, our idea to genetically engineer a phage which could potentially protect patients from extensive LPS release, seemed like a promising approach to her. However, she looked also at it with a critical eye and challenged us with a variety of questions that showed us the tremendous importance of thinking about every possible aspect when developing a therapy. We are very thankful for her insights!
In the very beginning of our talk she brought to our attention that a clinical study with phages can only be possible in combination with antibiotic treatment. The sole administration of phages as a therapy to patients with life-threatening infections would be considered unethical since antibiotics are still the best known method of treatment today. Thus, she could imagine PHANGEL as a co-treatment for patients struggling to recover and needing support by an additional antimicrobial agent which is simultaneously neutralizing the released LPS. Beyond that PHANGEL could also help in cases where no antibiotic is effective anymore due to bacterial resistances.
In this context she reminded us that not all antibiotics cause the destruction of bacterial cell wall and subsequent LPS release. This fact was important food for thought when thinking about the proposed implementation of PHANGEL. Combating LPS release is, therefore, only meaningful when cell lysis is taking place which is only the case for some classes of antibiotics.
Bacterial resistance mechanisms are not only a threat for antibiotic therapy but also for phages. However, the fact that bacteria seem to regain sensitivity against antibiotics when in contact with phages also underlines the usefulness of a combination therapy of antibiotics and phages. She also explained that therapeutic phage cocktails are applied to circumvent the problem of bacterial resistance. If a bacterium is resistant to one specific phage, another phage can still be effective. For exploring possible resistance formations, it could be interesting to know where exactly the T7 phage binds to E.coli.
Initially, PHANGEL aimed to be a treatment for older and immune deprived patients. Dr. Wienhold told us that genetically modified phages (or any therapeutic GMO) would probably not be a primary option for these risk patients since it is not sufficiently studied yet how the immune system would react to the engineered phages. The lack of acceptance for GMOs especially among older people could also pose a problem.
Since we aim to combat LPS released during bacterial lysis we should aim for an i.v. application and administer our drug directly into the bloodstream. In this regard, Dr. Wienhold stressed the very high purity needed for this and that the purification would be costly and complex. Several purification and buffering steps would be necessary.
In this context, she also raised our awareness that it must be distinguished between sepsis and bacteremia. Our approach would be more suitable to tackle bacteremia rather than the systemic state of emergency “sepsis”. However, in both cases, a timely start of the treatment is the most important thing.
She agreed that our aim to make the phages incapable of self-replication could make a potential approval process easier. However then high doses and possibly multiple rounds of application would be necessary for successful treatment.
Regarding our question how phages could survive the acid environment of the stomach we learned that usually antacids are given to neutralize the gastric acid before phage application. Our initial suggestion to coat the phages to protect them against acid would be rather impractical.
We also talked about meaningful questions we should ask ourselves for the further development of PHANGEL. Dr. Wienhold gave us some new impulses for our next steps. To test whether the released LPS would in fact be neutralized and thus rendered harmless by the immune system we could perform in vitro tests with macrophages and/or neutrophils. Other questions we need to ask ourselves now are:
How does the immune system react?
Which E.coli strains exactly are lysed by our phages?
How do we find our phages within the organism once they are administered?
She also pointed out that the phage titer could change over time and by this PHANGEL could lose its efficacy. Another thing we should consider is that the production strains should be tested for prophages.
We are very grateful for this highly informative discussion! Thank you Dr. Wienhold!
How we implemented the feedback in our project design
(Integrated Human Practices)
As mentioned above, the perceived tradeoff between an easier dosing regimen and our safety mechanism was an aspect in our project design that we discussed a lot after our talk with Alexander Belcredi. Our approach is only useful when our expressed protein gelsolin or the peptides GSN 160-169 and Peptide 19-2.5 achieve the elimination of a significant amount of LPS from the patients’ system. This task could be a lot more complicated to fulfill if the phages cannot adapt their concentration to the bacterial concentration through replication but have to be administered from the outside.
Sufficient bioavailability at the site of infection becomes a priority. Finally, we decided to keep our safety mechanism as planned because beside this disadvantage it also possesses the above mentioned advantages:
It is hard to disable this safety measure through mutations of the phage genome because we think it is unlikely that our transgene for gelsolin will mutate into major capsid protein within one generation and thus enable the phage to self-replicate.
It is a safety measure that can easily be transferred to other kinds of phages.
It facilitates a potential admission as a therapy because it cannot self-replicate.
It can be applied in a passive treatment regime which is not uncommon for phages.
Beside the scientific facts, another argument to have a non-replication phage is its acceptance in society and among the patients. When we asked our audience of the “Get Cozy with PHANGEL” talk whether they would accept a medical procedure with a GMO, the answers were mixed and only a few people completely agreed. An even smaller portion would accept the application with a self-replication GMO. Someone even confided with us that they thought the idea was scary.
Legal regulations might see this similarly: The microbiologist explained that it might be a difficult way until a genetically modified phage gets approved for medical use and in our talk with Dr. Wienhold we learned that the chances might be higher with an non-replicating GMO.
The talks also redefined our target group: we originally thought that this therapy would be relevant for old and immune suppressed patients as they are most likely to develop a sepsis in the first place.5 However, we learned that this group is also least likely to receive a not approved or GMO treatment. Hence, we reconsidered and target all ages now.
Furthermore, PHANGEL is unlikely to become a stand-alone therapy in the near future hence we reframed it as co-therapy to antibiotic therapies. This is due to two reasons: First, designing clinical trials for phage therapy is difficult and many ethical aspects need to be considered.The sole administration of phages as therapy for patients with life-threatening infections would be considered unethical: how would the control group be treated to make it comparable? Would they get no treatment? Antibiotics are still the best known method of treatment today and could only be complemented by the phage therapy. Furthermore, it has been shown that the interaction with bacteriophages can make drug-resistant bacteria more susceptible to antibiotics again.7 Considering these facts, PHANGEL can act as a co-treatment for patients struggling to recover and needing support by an additional antimicrobial agent which is simultaneously neutralizing the released LPS.
We also thought an oral application of the phages would be the easiest form of application. Even though phages cannot survive a low pH, we thought antacids or a coating could help them survive the stomach acid. This is indeed true, however, for patients with a sepsis it is not the most practical form of application. For these patients that are often unconscious an intravenous injection is the right way. This facilitates also the control over the bioavailability of the applied phages and spares pH stabilisation.
Because the phages are amplified in their production strain that is the same species as the infectious host strain, a lot of LPS is produced during phage production. To be able to inject the phages into patients, they need to be extremely pure.
Furthermore, for a continuation of this project, we would need to select another strain as producer as E.coli BL21 (DE3) contains a prophage which are legally not allowed for the production of therapeutic phages.
As you can see, the feedback from our experts shaped our project a lot. To find out more about the application of PHANGEL, visit our page about the proposed implementation.
1 Romero-Calle, D., Guimarães Benevides, R., Góes-Neto, A., & Billington, C. (2019). Bacteriophages as Alternatives to Antibiotics in Clinical Care. Antibiotics (Basel, Switzerland), 8(3), 138. doi: 10.3390/antibiotics8030138.
2 Guo, J., Chang, G., Zhang, K., Xu, L., Jin, D., Bilal, M. S., & Shen, X. (2017). Rumen-derived lipopolysaccharide provoked inflammatory injury in the liver of dairy cows fed a high-concentrate diet. Oncotarget, 8(29), 46769–46780. doi: 10.18632/oncotarget.18151.
3 https://www2.biomin.net/at/artikel/the-hidden-dangers-of-lipopolysaccharides/ (last seen on 26.10.2020).
4 Dedrick, R.M., Guerrero-Bustamante, C.A., Garlena, R.A. et al. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med 25, 730–733 (2019). doi: 10.1038/s41591-019-0437-z.
5 Starr, M. E., & Saito, H. (2014). Sepsis in old age: review of human and animal studies. Aging and disease, 5(2), 126–136. doi: 10.14336/AD.2014.0500126.
6 KERBY GP, GOWDY RA, et al. Purification pH stability and sedimentation properties of the T7 bacteriophage of Escherichia coli. J Immunol. 1949 Sep;63(1):93-107. PMID: 18139410.
7 Chan, B., Sistrom, M., Wertz, J. et al. Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa. Sci Rep 6, 26717 (2016).doi: 10.1038/srep26717.
