(INTEGRATED) HUMAN PRACTICES
Science is, and should be treated as, a social affair.
Human practice is an integral part of iGEM that attempts to map the societal and ethical implications of a scientific project. This is done by reflecting rigorously on how our project interacts with the world and by identifying stakeholders relevant to our project and investigating their values, norms and needs, on which we based design requirements for PGasus.
To assess whether we can really benefit the people and the environment, we have put a lot of effort into engaging many relevant stakeholders in the progress of our project and we integrated relevant stakeholder input into our reflections and our execution of the project. Not only the local social and economic context, where we aimed to innovate, was considered, but also ethical, legal, biosafety and biosecurity factors were thoroughly investigated by talking to experts. An elaborate overview of the project had been our greatest goal because, most importantly, our solution was designed to be safe to humans and the environment.
Human practice work is important for evaluating the viability of the project, and for evaluating where we can make the most meaningful impact. Here, we will show you how Human practice has shaped PGasus.
According to the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), genotyping tests must be implemented before prescribing certain drugs that are metabolized by CYP2D6 and CYP2C19 in order to recommend a dosage adjustment. However, some patients still do not respond to the treatment’s adjustment as expected, noting drug ineffectiveness and adverse drug reactions (ADRs).
Our team, in order to fully understand and address this problem, designed the following Human Practices pathway:
- UNDERSTAND THE PROBLEM
- Verify need
- Define a good solution
- Ethical & Application Concerns
Understand the problem
Being curious after receiving this information, we wanted to investigate it more to face these unexpected problems. After research we found out that according to the U.S. Food and Drug Administration (FDA) 2,216,000 serious Adverse Drug Reactions occur in hospitalized patients, causing over 106,000 deaths annually; thus side effects of medicines appear as the 3rd or 4th cause of death in the US(depending on the year). It is also well known that many medicines are more likely to be prescribed based on the patient’s genetic background. This procedure is explained by Pharmacogenomics, a new scientific field that describes the differential side effects in patients who receive the same dosage of a drug owing to differences in their genomic profile.
Thus, we decided to visit Dr. Patrinos’ office, professor of Pharmacogenomics and Pharmaceutical Biotechnology at the University of Patras and Head of the laboratory of Pharmacogenomics and Individualized Therapy, to find out what obstacles must be overcome to achieve the ideal personalized treatment.
He explained to us firstly that there are approximately 231 pharmacogenes, 50 of which regulate the CYP450 enzyme expression. He also highlighted that there are 40.6 pharmacogene variants and 10% of them are rare. These variants are significant for the general diversity of drug responses either these are side effects or incomplete pharmacological effect.
Approaching the science behind the problem
In order to investigate it deeper, Prof. Patrinos suggested reached out to experts in the field of Pharmacogenomics, Dr. Daniel Mueller and Prof. Sotiria Mpoukouvala.
Dr. Daniel Mueller is Head of the Pharmacogenetics Research Clinic at the Campbell Family Mental Health Research Institute at CAMH, and Full Professor in the Department of Psychiatry at the University of Toronto. Our conversation with Dr. Daniel Mueller was very enlightening. Not only he verified that even though many patients undergo pharmacogenomic testing, they eventually experience side effects, but he also gave us a greater picture of an issue we ignored.
The science of Pharmacogenomics is not yet well known and, in order to be further developed, it has to be accepted by the public; patients, doctors, pharmacists, government associations. We also discussed together about the procedure of detecting variants and making pharmacogenomics accessible to the patients. He told us that to perform an NGS analysis, he uses an accurate but time and money-consuming method. Thus, we are interested to find more efficient way to perform an NGS analysis.
However, we still needed more information upon the problem and that came from our conversation with Pr. Sotiria Mpoukouvala who is Associate Professor in Molecular Genetics, Director at Laboratory of Pharmacogenomics-Toxicogenomics at Department of Molecular Biology and Genetic at Democritus University of Thrace. She emphasized two important liver enzymes, CYP2D6 and CYP2C19, as they play a significant role in the metabolism of many prescribed drugs.
According to the European Medicines Agency (EMA), genotyping tests must be implemented before prescribing certain drugs that are metabolized by CYP2D6 (metabolizes 25% of clinically used drugs, Opioid etc.) and CYP2C19 (10% of clinically used drugs, antiplatelet etc.). These 2 genes are highly polymorphic, and this genetic variation is associated with profound changes in enzymatic activity. Mutations on these genes are responsible for the lack of efficacy and adverse effects of drug therapies, she highlighted. She also suggested we search in databases, such as Pharm Var, Pharmacogenomics knowledge base, Pharmacogenomics biomarkers in order to find exactly what we need to start our laboratory procedures. These databases helped us a lot for taking useful information and classifying the reported variants.
After all, we had fully understood the problem and we needed to verify the need for novel, rare variants to be detected. The first group we targeted was doctors. We believe that it is very important to know whether they are familiar with Pharmacogenomics and if they use it in their clinical workflow.
Dr. Mueller suggested we work with psychiatric drugs, such as antidepressants and antipsychotics which are metabolised by CYP2D6. He underlined that there are many people who suffer from psychiatric diseases and face problems with the drug dosage. Indeed, as psychiatric diseases are severe enough and difficult to treat, we agreed to the importance of finding the proper drug dosage to these patients.
Dr. Sotiris Patsilinakos (Medical Director of the Cardiological Department of Konstantopoulio General Hospital of Nea Ionia, Athens, Greece) confirmed the importance of the CYP2D6 and CYP2C19 enzymes and suggested that we did some research upon clopidogrel, an antiplatelet agent. His experience with a patient that almost died due to the unpredicted side effects of clopidogrel upon her was a trigger for us to search what variants of these enzymes may be the cause of it. He assured us that pharmacogenetic testing is a necessary tool and he would absolutely use it.
Dr. Konstantinos Nikitidis has been using pharmacogenetic testing for the last 2 years and believes it is a very powerful tool. He mentioned that he sends the sample from the patient to another country and the result is sent to him 2-3 weeks after. He made it clear that a test that gives results more quickly and is more affordable would be much more useful. He, also, listed a number of drugs that he thinks we should take under consideration, such as clopidogrel and opioid analgesics. After all, we had to find out what drugs we will use to perform our experiments, in order to investigate the CYP2D6 and CYP2C19 enzyme’s activity. We discussed it with prof. Patrinos and he proposed us to find it in dextromethorphan and mephenytoin. Although these drugs haven’t had a high impact in society, there are prototypes of drugs for conducting such experiments. We decided to use them in order to validate enzyme’s activity and then we will check our workflow in drugs proposed, too.
Our next step was to speak to a clinical pharmacist who comes across many cases everyday and deals with patients who experience side effects because of drugs. Mr. Diamantis Klimentidis a clinical pharmacist in the greek public hospital “Agia Aikaterini. He told us that genotyping tests would be beneficial in many cases and especially in elderly patients. He highlighted the importance of convincing doctors about the value of pharmacogenomic testing and suggested we present our project in clinics and doctors. Another issue that he raised was bioethics and that we had to be very careful as we are dealing with genetic information. He made us aware of the existence of the Laboratory of the Research of Medical Law and Bioethics in A.U.Th School of Law.
Presentation in Clinics and local doctors
After Mr. Klimentidis' proposal to do a presentation in clinics, we approached the hematology clinic of the General University Hospital of Patras, as well as a psychiatrist of the Social Insurance Institute. Prof. Argyris Symeonidis, who is the director of the hematology clinic, reassured us about the importance of making Pharmacogenomics more known, and explained why it is not widely applied. One of the problems he mentioned was the need for quicker and automized results. If we could overcome this barrier, we would make Pharmacogenomics more applicable.
Dr. Paraskevi Panagopoulou agreed that the personalization of the dosage is necessary in psychiatry since there are a lot of drugs that are metabolized by the enzymes that we investigated. She agreed that Pharmacogenomics could be a valuable tool in psychiatry because there are difficult disorders without an excellent existing treatment.
Finally, we had verified the need by people who deal with patients, but we would like to ask the opinion of patients themselves. As it was difficult to find patients to speak to, we decided to create an anonymous questionnaire in order to investigate and determine if the general public is familiar with the term “Pharmacogenomics” and to what extent. The results extracted show that a significant amount of the public is not aware of Pharmacogenomics and haven’t undergone a pharmacogenetic test even if there is a recommendation for the medicine they are taking. Most of the drugs they use fall into the categories of cardiovascular, psychiatric and oncological drugs as well as proton pump inhibitors, which show a significant amount of side effects. When asked if they would have such a test in the future, most of them answered positively. There were a few that weren’t sure mainly because they hadn’t met any doctor to recommend such an examination. Convincing doctors, the actual users of our device, appeared once again to be an issue to be dealt with.
At this point we knew we had to address this problem; all the possible variants of the CYP2D6 and CYP2C19 liver enzymes that contribute to side effects of cardiovascular, psychiatric, proton pump inhibitors and opioid analgesics had to be detected. We knew that synthetic biology would be a useful tool, as most of the techniques that we will use apply to synthetic biology.
Synthetic Biology as a solution
Dr. Koromina is a Post-doctoral Researcher in Bioinformatics and Statistical Genetics in the Laboratory of Pharmacogenomics and Individualised Therapy, School of Pharmacy, University of Patras. She discussed with our team the way we could identify the PGx variants of CYP2C19 and CYP2D6 as Prof.Sotiria Mpoukouvala proposed to us. As she mentioned, Next Generation Sequencing is the perfect approach for our purpose, as we can have high-throughput and targeted whole pharmacogene sequencing. She, also, underlined that through this approach we could detect common, rare and novel variants. By including a number of rare variants we ensure that some of them will appear in the population. She, also, suggested we include common variants as well. Finally, she made a comparison between NGS and the Sanger method; by using NGS we can be more confident about the result and it is economically advantageous when targeted sequencing is the goal.
When we were at a critical point trying to decide what device to use for sequencing, the answer came very unexpectedly when Mr. Anastasios Galanis, who is a Scientist in Oxford Nanopore Technologies, approached us. After hearing about our project, he recommended that we use Oxford Nanopore MinION. It offers a portable, low-cost sequencing and real-time sequencing data result and is just as accurate as the other NGS sequencers.
Following our conversation with Ms. Georgia Papanikolaou who is a PhD Candidate at the Department of Molecular Biology and Genetics in Democritus University of Thrace, we knew for sure that Oxford Nanopore was the ideal NGS device for our project, as she compared all the existing NGS devices. We also discussed together how important Pharmacogenomics is in clinical practice and how important it is to make research in it and detect more and more variants and their consequences.
Third-generation sequencing is progressing rapidly, moving from a technology once only capable of providing data for small genome analysis or performing targeted screening to one that promises high quality de novo assembly and structural variation detection for human-sized genomes. Oxford Nanopore MinION sequencer represents an exciting third-generation sequencing paradigm. MinION is a pocket-sized, portable sequencing device using novel nanopore technology. It contains an array of tiny holes called nanopores, embedded in a membrane, across which an electrical current is passed, and single-stranded DNA or RNA can be streamed through the pores and sequenced in real-time. This makes nanopore sequencing unique, it is the only sequencing technology that enables direct, real-time analysis of short to ultra-long fragments of DNA or RNA in fully scalable formats.
Processing to variant calling, it was noticed that among the already reported variants according to the genomic databases, some single nucleotide variants are not included in these databases and are supported by acceptable quality scores.
Along with Prof. Patrinos, we decided to create a comprehensive workflow for the identification & functional characterization of Pharmacogenomics variants. Prof. Patrinos, informed us that early pharmacogenomic platforms were confined to the analysis of common variants. “Consequently, there may be unreported, low-frequency variants, called novel variants, the effects of which are significant enough to be considered.” as he highlighted us.
However, we had to determine whether the novel variants were damaging or not for the function of the enzymes needed for the metabolism of the drugs’ prescribed in order to understand their importance and make Project PGasus an accurate and repeatable project.
Experts in the Wet Lab
But how could this be possible? We knocked on Dr. Diana Portan’s office, who is a researcher in CCAMF-UMFST, Tg. Mures in Romania and an Academic Fellow at Mech. Eng. & Aeronautics Department in University of Patras, to find the answer. She told us that we had 2 options. Either we should use the CRISPR-Cas9 system or site-directed mutagenesis in eukaryotic cells.
Prof. Argyro Sgourou who is an Assistant Professor at School of Science and Technology in Hellenic Open University, was very helpful by providing us with the necessary knowledge concerning the characterization of potentially damaging consequences of novel variants. She suggested performing in vitro assays in eukaryotic cells to examine the expression of CYP2C19 and CYP2D6 variants. We conducted site-directed mutagenesis in 293FT cell line. The 293FT Cell Line is a fast-growing, highly transfectable clonal isolate derived from human embryonic kidney cells transformed with the SV40 large T antigen, allowing very high levels of protein to be expressed from vectors containing the SV40 origin.
After all, we had to find out what drugs we will use to perform our experiments, in order to investigate the CYP2D6 and CYP2C19 enzyme’s activity. We discussed it with prof. Patrinos and he proposed us to find it in dextromethorphan and mephenytoin. Although these drugs haven’t had a high impact in society, there are prototypes of drugs for conducting such experiments. We decided to use them in order to validate enzyme’s activity and then we will check our workflow in drugs proposed, too.
Finally, to determine the enzymes’ activity, we collaborated with another laboratory. Specifically, the cells containing wild-type and CYP2D6 variants were incubated in a mixture containing substrate dextromethorphan, and cells containing wild-type and CYP2C19 variants were incubated in a mixture containing mephenytoin. The determination of dextromethorphan in the microsomal protein fractions was measured with Ultra-Performance Liquid Chromatography (UPLC) - Fluorescence (FLR) detector system and for CYP2C19 with Liquid Chromatography tandem Mass Spectrometry (LC/MS-MS).
Our in-vitro findings validate the initial assumption of the in silico score regarding the damaging effect of the novel variants. Τhe effects of these novel variants are significant enough to be considered in determining potential clinical actionability even for drugs with established genetic evidence-based dosing adjustment.
After a conversation with Jessica Martyn, our iGEM mentor, we took our project to a new level. She suggested that we have to find a way to use modeling to gain insight into the interaction of the compound drug and the catalytic center of the enzymes of interest - mutated with the already discovered novel variants.
Our conversation with Ms. Athina Eleutheraki who does MSc in Toxicology at Karolinska Institutet and was iGEM Stockholm 2019 team Leader helped us to achieve our goal. She is also a former iGEMer, so we had to discuss a lot with her. After she gave us a lot of information about iGEM competition and advised us in order to succeed, she suggested that the Docking procedure was good for modeling our project. In particular, 3 Dimensional Structural Modelling was helpful to observe and predict the interactions between recombinant enzymes and drug substrates.
Last but not least, we approached Prof Vizirianakis, who is an Assistant Professor of Molecular Pharmacology and Pharmacogenomics at the Department of Pharmaceutical Sciences in AUTH, because we were searching for an alternative way to evaluate the CYP2D6 enzyme expression. According to past studies we find out that MicroRNA hsa-miR-370-3p suppresses the expression and induction of CYP2D6. We agreed that the Toehold Switch could be useful for the detection of this microRNA. So, our fellow team iGEM Thessaloniki team designed for us the appropriate toehold switch sequence.
Nearly every single person we talked to, raised their concerns about the bioethics of our project. For this reason, we decided to get in touch with the Laboratory of the Research of Medical Law and Bioethics in A.U.Th School of Law, as suggested by Mr. Klimentidis. It was important for us to know exactly what are the legal and ethical issues that come along with a project like ours and how we can prevent them. Their answer was as follows: “The issue of detailed information of the participants about the process and the content of the project, as well as its future extensions, is crucial for obtaining their valid consent. The informed consent of the participants in such projects is a necessary condition.”
The issue of detailed information of the participants about the process and the content of the project and its future extensions is crucial for obtaining their valid consent. The informed consent of the participants in such projects is a condition, of course. For this reason, applying for approval of the program to the Ethics and Ethics Committee of the University and obtaining approval from it ensures that the legal procedures have been followed and all the prescribed conditions have been met. Moreover, the strict application of what is stated in the documents for approval of the study is a guarantee of the prevention of ethical issues. It should not be forgotten that research should be conducted with respect for human dignity, as protected by guidelines, international conventions, and declarations (such as the UNESCO Universal Declaration of Bioethics and Human Rights, the UN Convention on the Elimination of All Forms of Discrimination against Women). Finally, researchers must adhere to the professional ethics of their field when conducting research of that type.
Besides the ethical aspect of our project, we needed to discover its cost-effectiveness. Ms. Christina Mitropoulou who is a Managing Director and Senior Health Economist at The Golden Helix Foundation, analyzed the potential economic problems and the economic policy to be followed. She made us consider the value of the novel variants that were detected and whether this diagnostic workflow would be beneficial for its users. We also discussed the potential NGS sequencers we could use, and she agreed that Oxford Nanopore would be ideal as it is cost-saving and just as accurate as the other NGS sequencers.
At this point, we started to get curious whether the diagnostic tool we were designing could be approved by government associations. For that reason, we approached the National Organization for Medicines and the European Medicines Agency(EMA). Our discussion with the National Organization for Medicines was about the process for a device to be approved as a diagnostic tool and how we could make it suitable for approval. Ms Virginia Safra, who is an employee in the Department of Medical devices at the National Organization for Medicines, gave us advice concerning the steps that have to be made for a sequencer like Oxford Nanopore MinION to be clinical approved for the targeted sequencing of two pharmacogenes and the information amused us. Moreover, we got in contact with Mr Iordanis Gravanis, Head of Office in the Scientific Advice and Scientific Evidence Generation Department at the European Medicines Agency, who highlighted that any innovative technology related to drug development is positively examined to get approval. Although our project doesn’t concern drug development, it deals with the improvement of drug administration, something crucial for many patients. A cost-saving and easy tool that helps diagnostics laboratories and doctors would be very beneficial. Thus, Mr Iordanis from EMA urged us to think of ways that could make our device attractive and valuable for adopting them in clinical practice.
At last, a company that deals with genetic tests, Genekor, approached us in order to find out more about our diagnostic tool. Our conversation gave us important information about the possible future steps for promoting our project, its advantages and how to convince its potential users to get advance of it.
Our proposed solution
After all, our complete proposal for addressing this problem had been formed by taking into account all of the advice we had throughout our human practices pathway.
Except for Genekor’s help regarding the future steps we have to make in order to promote our project, they informed us about the fact that the comprehensive workflow for identifying PGx variants that our team proposes requires knowledge of NGS analysis results that a physician or a doctor cannot do. The second time we reached all the doctors we had contacted during our Human Practices - when our project was fully developed - they confirmed Genekor’s point of view. So, we had to consider diagnostic laboratories of either public or private clinics or companies as the primary end-users of our project. In other words, experts in Bioinformatics placed in diagnostics laboratories could easily analyze the results derived from the NGS device. At this point, we must mention that although our team has already discovered its potentials as a diagnostic tool, Oxford Nanopore MinION should be approved by the FDA and other health regulators as a legitimate device capable of diagnostic procedures, as Ms. Virginia Safra, an employee in the Department of Medical Devices in our National Organisation for Medicines, informed us.
Solving the gap between science and the community
In order to establish our approach as an everyday diagnostic tool, due to its advantages, for public health systems around the world the main future step of our project is to develop a neural network for automatization of the NGS analysis, to make the results understandable by health professionals, in order to make the pharmacogenomics more easily implemented in clinical practice. This could be a total game changer for our project since doctors will be more likely to get advantage of this sequencer’s device, as Christina Mitropoulou highlighted. Last but not least, we hope to collaborate with the Ministry of Health to make our workflow adaptable to health-care units while insurance companies could cover the operation cost. Being affordable, time efficient, and easily transported to remote districts are the key points to persuade the government for its implementation.
As far as the detection of the novel variants is concerned, after acquiring the knowledge that some of them have damaging effects on the CYP2D6 and CYP2C19 enzymes' activity, after the functional characterization procedures we conducted, we propose that they should be included in research studies and clinical trials. This is a crucial step to be made; since then, we will be able to validate the exact dose that a patient with such variants should be administered. This is a long-lasting procedure because the different disorders in the patients' physiology, such as cardiopathies or nephropathies, must be considered, as Mr. Vizirianakis mentioned. Moreover, for once more, the Food and Drug Administration (FDA) must approve the clinical trials conducted upon these variants and the proper dosage administration of the drugs metabolized by the enzymes of interest, as Mr. Klimentidis informed us. After the FDA approval, lab technicians in diagnostic labs, for starters and later doctors in clinics, will have the prospect to also consider these variants before prescribing a drug to a patient by adjusting its dose.
Using Pharmacogenomics to minimize the ADRs of a wide range of drugs and the subsequent deaths and to enhance the quality of life for the patient has been our duty for the last ten months. This will result in the ideal personalized treatment and in redefining 21st century Medicine.