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Human Practices

Cancer remains one of the most deadly diseases, killing a large number of people in the world each year. Its survival rates depend highly on both criteria: the detection time and the efficacy of the treatment. However the second one is itself highly influenced by the first one. Consequently, even if a lot of cancer biosensors exist already it is important for us to increase the accuracy and the fast-detection of cancers. In this project, we want to improve early-stage cancer detection using a new kind of biosensor that is more accurate than the ones producing visible or fluorescence output. We create a biosensor with an electrical output and measure the output using either a multimeter or an embedded Arduino chip device, thus improving sensitivity. By using inexpensive and accessible technology, we hope to improve early cancer diagnosis in order to improve clinical outcomes for cancer patients.

The design of our project was influenced by all the people we discussed with: scientists, cancer patients, a bioethics professor, a lawyer, pharmacists and analysis laboratories. It's thanks to all of them that we know on one hand what we are allowed to do, what patients are expecting from the test and what is feasible taking into consideration those two previous aspects. By making an extensive bibliographic work, and meeting with cancer patients and researchers allowed us to confront the reality behind the disease on both social and scientific levels. We asked cancer patients about their stories, how they found out about the disease, and how much time it took them to process that they were in danger. In addition, we spoke to researchers and they told us what part is feasible in our project and what part needs to be modified to fit our goals.

Thanks to various papers from different researchers, the establishment of both iGEM check-in and safety forms and the testimony of a former cancer patient, we had the evidence that our project did not represent any kind of risk towards both our environment and the humans and could represent a great device for early-stage cancer detection.

All of these discussions inspired us and motivated us to work more on this biosensor.

Scientific feasibility

To better evaluate the feasibility of our project from a scientific point of view, we contacted researcers specialists in external electron transfer. We are greatfull to Dr. Ricardo Louro, Dr. Jeffrey Gralnick and Dr. Benjamin Keitz who kindly listened to us and shared their knowledge.

We presented them our idea of modulating the external electron transfer in our engineered MtcCAB and Cytochrome A (CymA) Escherichia coli through the expression variability of the CymA nanoconduct depending on the presence of our lncRNA PANTR1 120 base-pair sequence. They found it very original because it was the first time they heard of such an application for a microbial fuel cell, and they were very interested in our project. According to them, the common use of microbial fuel cells is more wastewater treatment oriented.

What we learnt from these interviews is how important are both the riboflavin and the cytochrome A (CymA) nanoconduct compared to these MtrCAB nanoconduct (that remain according to them very unspecific for each bacteria, that is the reason why one can implement them in a considerable number of microorganisms). According to them, the use of CymA as a regulator of external electron transfer was a really good idea. However, they warned us about a certain aspect of our project we might struggle with. Even if the MtrCAB nanoconduct remains unspecified and can be implemented in different bacteria, the incorporation of such a complex membrane protein is very time-consuming in E. coli, and we might not have proper results in a few months of work because of that.

They advised us to just use the CymA toehold switch in E. coli and to remove the MtrCAB genes plasmid. Although we would have a great electric signal lost, as we only need to get a significant electric difference between the ON and the OFF state, the power of the electric current produced does not matter.

However, we decided to try incorporating the MtrCAB genes in order to optimize our current production, but faced E. coli growth problems. In parallel, we performed experiments taking into acount this advice, but the presence of only CymA did not lead to a detectable increase in the electrical output.

Patient Testimony

We also needed the opinion of cancer patients to enlighten the social view of our project. A former cancer patient accepted to answer our questions related to how he discovered he had cancer and the entire medical journey he went through in order to know that he had this disease.

"It is by chance that I learnt that I had cancer, it began when I went to an appointment with my regular doctor who detected a nodule in my throat. I got a puncture to check the size of the nodule.
According to the radiologist, the nodule was not malignant, my worrying decreased significantly. However, after the puncture, I felt a terrible persistent pain in my throat and it was getting worse each day.
I went to an endocrinologist's office and he said to me that it was just an inflammatory problem, nothing very terrifying according to him.
3 months later, the pain remained and was really difficult to endure. I felt pain from the bottom of my neck to my ears. This is why I looked for an ENT (otorhinolaryngology) specialist, who saw that something more serious was wrong with me and advised me to get a surgical operation.
This first operation was really complicated because the nodule was bound to other organs, but as it was not a malignant nodule (according to doctors) there was no need to remove it.
A month after the first operation, I always felt the same amount of pain, so I decided to get a second opinion at a different hospital. I was rapidly taken in charge and the nodule was classified as malignant. So, this is how I learnt I had cancer.”


What we can learn from this testimony is that human error seemed to be the major problem this person faced. It may be an isolated case. However, the genetic test seems to be more appropriate. Our test is based on RNA complementary base pairs binding, with a strong correlation bound between A/T and G/C. Moreover, since the test is relatively non-invasive one can repeat it multiple times in case of doubt.

Safety and Security

This cancer patient opinion is not the only one that matters. Indeed, regarding the law (see bellow), we have the obligation to give detailed explanations on how our device is working in order for the society not to be afraid of our project just because we used engineered Escherichia coli.

E. coli does not have a good public image. For instance, this year, a scandal occurred in France due to a famous brand of pizza that was not fitting the sanitary standards and a lot of people got contaminated by E. coli (according to France public health, 56 peoples were poisoned and 2 of them died due to hemolytic and uremic syndromes). As a consequence, when we discussed our project with our own acquaintance, we found that this case deeply affected their mind and for them E. coli was a dangerous bacteria (while it is not in most cases and is one of most known organisms and the most used in synthetic biology research). Adding to this the fact that we were engineering this same bacteria to make it a GMO seemed purely dangerous for them. That is why we will need to inform people on E. coli before we start human blood sample tests in order to reassure our community and the whole society about E. coli.

The main issue from a Safety and Security point of view of our project was the use of the PANTR1 long non coding (lnc) RNA. lncRNAs are regulators of gene expression at epigenetic transcriptional and post-transcriptional levels [1]. PANTR1 is overexpressed in different types of cancer cells and it was shown to promote cell migration, invasion, angiogenesis and proliferation and to repress apoptosis [2-9]. In the beginning, we wanted to design a toehold switch and use as trigger the full length PANTR1 lncRNA sequence to mimic the natural samples. However working with the whole sequence might be dangerous for the human beings. To take into account this risk, we modified our project and only used shorted fragments of 120 base-pairs that does not represent a danger. This choice was confirmed by the iGEM Safety and Security committee that validated both our Safety and Check-in forms.

Legal aspects

One of the main point we wanted to be enlightened with was the law one. Laws result from experience, knowledge, and the mistakes of our predecessors. To integrate it in our project, we needed more informations about how the legal system is considering the human blood and tissues samples, genetic tests, the use of GMOs and what we are allowed to do or not to do. Thanks to Adv. Karine Chrunyk we obtained informations about the law texts used as a reference by both lawyers and magistrates of the Bobigny (France) law court.

Research actors on the human being

There are different kinds of actors in the research in the human beings area. The promoter names a coordinator investigator in charge of a research located in several sites in France (L. 1121-1 article of the French public health code).

The national research commission involving the human persons is ruled by the health ministry (L. 1123-1-1 article of the French public health code). It is fulfilling several missions:
The protection committee for peoples are structures formed with public rights people. If the committee makes a mistake, it is the French state that will be responsible for their mistake (L. 1123-7 article of the French public health code). They are working on their missions independently, so they must be composed of people with a wide skill range in the research on the human being domain, ethics, sociological, psychological and legal. Some of their members should be from an association defending sick people or from a user of a recognized health system (L. 1123-2 article of the French public health code).

The National Agency for the Safety of Medicines and Health Products (ANSM) participate in the application of all the rules related to research involving human beings (L. 5311-1 article of the French public health code). Its decisions in terms of research are either tacit autorisations (the research is accepted if no answer is sent back to you) or express autorisations (the research is not accepted if no answer is sent back to you). In our case, we are interested in express autorisations, as the research carrying on the following products needs an express autorisation delivered by the general director of the ANSM. Are concerned by the R. 1125-7 article of the French public health code: An express autorisation is mandatory for research including a medical intervention not justified by the common care of organs, tissues and human origin cells (L. 1125-7 article of the French public health code). The biomedicine agency delivers an opinion on research including a medical intervention not justified by the common care of organs, tissues and human origin cells. The general director of the biomedicine agency sent his opinion to the general director of the national agency for medication security and health products.

Genetic research laws

The first genetics laws appeared in France in 1994 in the French civil code. Since the 29th of July 1994 (Law n°94-653, Civil Code, 16 to 16-13 Articles, 10th Instalment) the civil code takes into acount two kinds of tendencies relative to human genetics.

First of all, the 16-4 article provides measures to protect the human kind from genetic applications. Those measures are developed in the 30th Instalment). This same article was updated by the 6th of August 2004 law relative to bioethics features (Law n°2004-800).

The second type of measures are described in 16-10 to 16-13 articles (several times modified since their creation). Those articles do not protect the human kind but more particularly the individual against a possible use of their genetic information for any illegal activity. For example, the 16-10 article says that an examination of genetic features cannot be allowed for purposes other than medical or scientific ones. The examination cannot be made without the consent of the person and even if the person gives it before the examination, the person can revoke the consent during any stage of the examination. Plus, the person has to be warned of the nature of the examination and the end of it.

In France, the protection of individual genetic data is strengthened by the 226-26 article of the penal code stipulating that the fact of diverting genetic data is punished by the law by a year of incarceration and a penalty of 15 000 euros. Medical purposes for genetic data are controlled too by the R. 1131-1 article of the French public health code. It embeds the fact that it is allowed to interrogate, affirm or deny the diagnosis of a genetic disease (as cancer for example). Such an examination could be conducted either on a symptomatic or asymptomatic patient.

Both laboratories and people making the diagnostic must receive an authorization. People making the diagnostic test receive an authorization from the general director of biomedicine agency according to the R. 1131-6 to R.1131-12 articles of the French public health code. Biological analysis laboratories are allowed to make those diagnoses thanks to the L. 1131-2-1 article of the French public health code since the law of the 7th July of 2011. Moreover, this autorisation could be revoked in cases of negligence toward genetics regulations and laws.

There are a lot of various national laws dealing with human genetics but there are European laws too. It is the case for example for the Oviedo convention that remains as a reference in the genetic domain. This convention is about both Human beings’ rights and biomedicine (Oviedo, 4th of April 1997), the European ministers committee ordered the current “Director committee for Bioethics” to write down this convention. It is composed of 38 articles and is linked to the European human rights convention made on the 4th of November 1950. Those two texts have in common to be both centered on ethics and law principles. The fundamental value defended by this convention is the human dignity one notably the 1 and 2 articles. Moreover, this convention treats inclusivity aspects too, it is the case for the 11th article where it is written, that all form of discrimination toward a person because of his genetic heritage is prohibited and punished by the law. In compliance with the 4th paragraph of the 33th article of the convention, the French government passed the convention on the 1st of April 2012.

The fundamental right charter of the European Union contains rules dealing with genetics too. The 3rd article forbids human cloning and eugenics to protect the human dignity and the 21th article forbids discrimination based on genetic heritage too.

UNESCO passed two rules too, the universal declaration on the Human genome and rights of the 11th of November 1997 and the international declaration on Human genetics data of the 16th of October 2003.

GMO

As we are working with engineered E. coli to make our project successful, we will have to work with a Genetically Modified Organism (GMO) and sell it on the market for our future implementation.

The GMOs that we want to use are regulated to the L.531-2, D.531-1 to D.531-3 articles of the environmental code which stiplulates that traditionally used techniques for genetic engineering that are known as safe for both the public health and the environment are listed by the biotechnologies high council. According to the L. 531-2-1 of the environmental code, introduced by the 25th June of 2008 n°2008-595 law, GMOs can be cultivated, used or commercialized only if they do not represent a danger for the environment, the public health, farming infrastructures, local ecosystem and both commercial and production industries displaying “without GMOs”. The “without GMOs” definition is given by the European definition. By waiting for a definition to the European level, the threshold is given by the biotechnologies high council (in France), species by species.

Autorisation decisions cannot be given before the independent and transparent assessment of the risk that a particular GMO can represent both for the environment and the public health.

The 25th June 2008 law created the biotechnologies high council to enlighten the government on all the questions concerning both GMOs and biotechnologies and to give assessment concerning the voluntary spreading or the confined utilization of GMOs in both the environment and the French public health code (according to the L. 531-3 article). This council creates assessment methods based on both community tendencies and international recommendations. According to the L. 531-4 to L. 531-5 and to the R. 531-7 to R. 531-28 articles of the environmental code, this high council is composed of a scientific committee, an economic committee, an ethics committee and a social committee. After the assessment of the scientific committee, both the social and economic committees devise recommendations and can afterwards summon both a member and the president of the scientific committee. The result of this meeting is given to the biotechnologies high council president, those results take into account risks and profit assessment and the difference of opinion between those committees (according to the L. 531-4 article of the environmental code).

According to some laws our GMOs cannot get a marketing authorization. For example, according to the L. 533-3, L. 533-5 and R. 333-1 to the R. 333-51 articles of the environmental code, marketing authorization cannot be delivered for GMOs containing antibiotics resistance genes used for either medical or veterinary treatments (such as ampicillin) for which the assessment of risk conclude that they could have an harmful effect on both the environment and the public health. Moreover, in our project we are using a resistance gene for ampicillin in our engineered E. coli secreting electron shuttles mediators, plus only medical analysis laboratories or a practicer who received an autorisation from the general director of the biomedicine agency are allowed to work with genetic test (cf. the genetic rules part).

So, we will have to pass through a restricted use for our test.

According to the L. 532-1 and the R. 532-1 to R. 532-4 articles, GMOs are classified into different groups depending on the risk they represent according to a rank fixed by decree after that the biotechnologies high council gave his opinion on a particular GMO. All use for teaching, research, industrial production of GMO that might represent a danger or a disadvantage for the environment or the public health must be done under confined conditions. The confinement is used to raise physical, chemical or biological barriers to avoid contact with the environment. After the biotechnologies high council had given his opinion on a particular GMO, the confined use authorization is given by administrative authority. The use can be submitted to a declaration only if it does not represent a risk or not an important one, it will take place in installation already accepted for organism ranked in the same or superior class (L. 531-4, L. 532-2 to L. 532-6 and R. 532-1 to R. 532-34 articles of the environmental code). If a new assessment of danger and disadvantages for both environment and public health is needed, regarding new data for example, the administrative authority can impose a dictate modification, suspend or revoke the autorisation (L. 532-5 article of the environmental code).

The fact of exploiting an installation using GMOs for research, development, teaching, or industrial production without the autorisation required by the L. 532-3 article of the environmental code, or breaking techniques dictate in whom the autorisation is subordinated, is a crime punished with a year of imprisonment and a 75 000 euros penalty. According to the L. 536-3 article of the environmental code, the court can shut down the installation functioning until a new instruction is given. The fact of exploiting such an installation breaking imposed dictate in application with the L. 532-5 article, or violating a suspension or revoke dictate measure is punished by 2 years of imprisonment and a 150 000 euros penalty, the court can decide to shut down the installation functioning too (L. 536-3 article of the environmental code).

The lack of dropping off of an information file in the municipality city hall or in the implantation district of the laboratory you are working in, in the anticipated conditions by the R. 532-13 article is punished by the law. According to the R. 536-11 of the environmental code, the fact of not dropping off such a file is considered a third-class minor offense when you are working with GMOs having a third or fourth confinement class (D. 532-3 article of the environmental code). The fact of not warning the minister in charge of the research that a laboratory accident lead to environmental or public health harmful consequences is considered as a fifth-class minor offense (R. 536-11 article of the environmental code). The responsible for the laboratory will be punished according to the R. 532-14, R. 532-14 and R. 536-11, III articles of the environmental code).

Blood and tissue sample

According to the L. 1243-2 article of the French public health code, only few medical establishments / organisms are allowed by both the biomedicine agency and the ANSM to prepare, keep, and distribute tissues for therapeutic ends. The autorisation is delivered by the ANSM, in conformity with the 6th article of the 2004/23/CE of the European parliament and with the 31th of March 2004 council related to creation of quality and security standards for donation, the obtainment, the control, the transformation, the preservation, the storage, and the distribution of both human cells and tissues. All modifications changing the initial autorisation without any autorisation is not allowed. If there is new data to take in account, the medical establishment or organism must obtain a new autorisation. Other modifications are directly submitted to the general director of ANSM.

Apart from application with a therapeutic end, the public health code takes into account the possibility of scientific activities (for diagnostic in our case for example). These organisms do not have to receive a specific autorisation but they have to submit a preliminary declaration. According to the L. 1243-3 article, they have to send it to the research ministry for their research programs needs. The preliminary declaration discusses the preservation, the preparation to scientific ends of cells or tissues coming from the human body and their respective own components and derived products. Those activities include both the formation and use of human biological sample collections. When it is a medical establishment making this type of declaration, it is sent directly to both the research minister and the general director of the health regional agency.

We hear by the expression “human biological sample collections”, the gathering, to scientific ends, of biological sampling done on a group of similar people and selected depending on their clinical or biological characteristics of one or several members of this same group. The ANSM is aware of all the establishment's actions related to blood and tissue sampling, if there is a problem with this organism, the ANSM can suspend those activities. Today, as with the other elements and products of the human body, both the donation and use of human blood are submitted to ethics order general principles given by the first title of the second book of the first part of the French public health code, by the civil code and by sanitary security requirements. The three essential principles of free, anonymity and consent of the donor are already written in the 1993 law, putting related rules to both sample and blood collection conditions (contained today in L. 1221-1 to L. 1221-14 articles of the French public health code). The blood sample is a breach of the physical integrity of the person herself, covered by the no-patrimonial principle of the human body, the blood can only be given for both conditions and ends defined by the law.

“A blood sample can be used for other purposes than the conservation and restoration of the health of someone: a sample can be used for diagnostics purposes, scientific research ends, as proof in both civil and penal right.” So, blood samples can be used either for medical or research purposes, which in our case is good news, as we enter in both categories. We will have to take blood samples from cancer patients in order to see if our diagnostic device is working with real blood containing the lncRNA PANTR1 or not. In the context of our project this means that we might have the autorisation to be recognized as a scientific research team and will not need a lot of autorisations to work with human blood to test if the lncRNA PANTR1 concentration in the blood of cancer patient is enough to detect an electrical output through our system.

Medical purposes for genetic data are controlled too by the R. 1131-1 article of the French public health code. It embeds the fact that it is allowed to interrogate, affirm or deny the diagnosis of a genetic disease (as cancer for example). Such an examination could be conducted either on a symptomatic or asymptomatic patient. Both laboratories and people making the diagnostic must receive an authorization. People making the diagnostic test receive an authorization from the general director of the biomedicine agency according to the R. 1131-6 to R.1131-12 articles of the French public health code.

Both the European and the French laws do not seem to have very specific requirements for genetic cancer detection using blood or tissue samples, but just a reference list of persons allowed by the biomedicine authority are allowed to practice such genetic tests. So, we will need to get such an autorisation for our preliminary blood test.

According to the L. 531-2-1 of the environmental code, introduced by the 25th June of 2008 n°2008-595 law, GMOs can be cultivated, used or commercialized only if they do not represent a danger for the environment, the public health, farming infrastructures, local ecosystem and both commercial and production industries displaying “without GMOs”.

Thanks to our previous work on both check-in and the safety forms, and with the help of a bioethic teacher, Prof. Thomas Heams and Prof. Fariza Tahi, a researcher in lncRNA, we found out how we might be able to limit the risk in the wetlab part of the iGEM competition. However, in the real-life implementation of our project we will have to be way more cautious because we will have to work not with a fragment of 121 base-pair-long lncRNA PANTR1 but with its whole sequence.

The autorisation delivered by the ANSM, in conformity with the 6th article of the 2004/23/CE of the European parliament and with the 31th March of 2004 council is related to creation of quality and security standards for donation, the obtainment, the control, the transformation, the preservation, the storage, and the distribution of both human cells and tissues. In order to obtain the autorisation we will have to show that we will be able to comply a lot of norms related to security standards, preservation and storage of human cells and tissues.

Medical professionals

Finally we searched for help, opinions, and advice from professionals that would be impacted by our project. For this we interviewed people who work as pharmacists and in medical analysis laboratories, who turned out they never heard of cancer biosensors or synthetic biology. We presented our thoughts to them about the implementation of our project. We wanted to give them either our engineered E. coli under lyophilized or frozen form, so that they would then perform either an electroporation or a heat-shock to allow PANTR1 lncRNA present in the blood or tissue sample in enter the cell. We would give them a copy of our hardware and instructions on how to use it.

However, people working in analysis laboratories were more suitable to perform such experiments, plus they might also have an appropriate freezer to keep our own engineered E. coli at low temperatures.

However, people working in medical imaging were skeptical regarding the way our engineered E. coli was working, so it might be a challenge to convince those laboratories to use our device.

In our opinion, people who could be directly affected by our project would most probably be the people working in medical analysis as they would have less work due to our device being in charge of the diagnostic process.

Moreover, if our project is well-implemented, both chemotherapy treatment and our diagnostic tool should be able to contribute toward a decreased cancer death rate due to earlier and better diagnostics, and this is what we aimed for.

References

[1] Vance KW, Ponting CP. Transcriptional regulatory functions of nuclear long noncoding RNAs. Trends in genetics: TIG (2014) 30: 348–355.
[2] Li W, Zheng J, Deng J, You Y, Wu H, Li N, Lu J, Zhou Y. Increased levels of the long intergenic non-protein coding RNA POU3F3 promote DNA methylation in esophageal squamous cell carcinoma cells. Gastroenterology (2014) 146: 1714-1726.e5.
[3] Guo H, Wu L, Yang Q, Ye M, Zhu X. Functional linc-POU3F3 is overexpressed and contributes to tumorigenesis in glioma. Gene (2015) 554: 114–119.
[4] Tong Y-S, Wang X-W, Zhou X-L, Liu Z-H, Yang T-X, Shi W-H, Xie H-W, Lv J, Wu Q-Q, Cao X-F. Identification of the long non-coding RNA POU3F3 in plasma as a novel biomarker for diagnosis of esophageal squamous cell carcinoma. Molecular Cancer (2015) 14: 3.
[5] Shan T-D, Xu J-H, Yu T, Li J-Y, Zhao L-N, Ouyang H, Luo S, Lu X-J, Huang C-Z, Lan Q-S, Zhong W, Chen Q-K. Knockdown of linc-POU3F3 suppresses the proliferation, apoptosis, and migration resistance of colorectal cancer. Oncotarget (2016) 7: 961–975.
[6] Lang H-L, Hu G-W, Chen Y, Liu Y, Tu W, Lu Y-M, Wu L, Xu G-H. Glioma cells promote angiogenesis through the release of exosomes containing long non-coding RNA POU3F3. European Review for Medical and Pharmacological Sciences (2017) 21: 959–972.
[7] Li Y, Li Y, Wang D, Meng Q. Linc-POU3F3 is overexpressed in hepatocellular carcinoma and regulates cell proliferation, migration and invasion. Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie (2018) 105: 683–689.
[8] Wan X, Xiang J, Zhang Q, Bian C. Long noncoding RNA POU3F3 promotes cancer cell proliferation in prostate carcinoma by upregulating rho-associated protein kinase 1. Journal of Cellular Biochemistry (2019) 120: 8195–8200.
[9] Li W, Wu X, She W. LncRNA POU3F3 promotes cancer cell migration and invasion in nasopharyngeal carcinoma by up-regulating TGF-β1. Bioscience Reports (2019) 39: BSR20181632.