Overview

Synthetic biology techniques as CRISPR-Cas9 have gained huge public interest for human enhancement and are becoming more and more accessible to the general public. In this light, we identified the need to promote responsible use of synthetic biology. The discussion on human enhancement takes a most prominent place in sports with the doping affairs and unites with synthetic biology in the phenomenon of gene doping, for which an implemented detection system lacked. Therefore, we decided to develop an efficient, reliable and versatile detection method for gene doping based on a thorough value sensitive design.
In the initial stages we presented our idea at the Bioengineering Institute Kickoff and immediately caught the interest of Clive Brown, Chief Technology Officer at Oxford Nanopore Technologies. Skype calls with the company ensued and drove us to switch our idea from a nanopore blocking to a pulling method.
Subsequently, we visited the VVBN conference on advances in doping to gain more insight in the field. Here, we met Dr. Dimeo, Professor in Sports Policy, who prompted us to extend our model to anticipate athletes’ choices in gene doping administration.
This idea to anticipate on future athlete behaviour also led us to organise the Hackathon at the Cyber Security Week. By letting engineers hack our detection method, we obtained additional variations for possible gene doping sequences, which were automatically added to our database.
Then, we presented our project for life science experts at the Delft Health Initiative where we discussed the impact of gene doping on the environment and future generations. This led us to involve a broader public through the organisation of the first Dutch Biotechnology Day characterised by debates we instigated on trains throughout The Netherlands.
However, we wanted to take it further and organised an expert discussion on the topic at the University of Stirling, Scotland’s University for Sporting Excellence. Here, we focussed on the differences between gene doping and more conventional doping in all aspects and how scientists should respond. Here, it became even more apparent how vulnerable athletes are to doping use and our approach to education was reinforced to close the loop for future responsible research.

Approach

As a team we highly value responsible research. Therefore, we wanted to make sure our project is responsible from the start till the end and beyond. This we made sure by passing our project through the phases that constitute Responsible Research and Innovation according to Stilgoe et al. (2013), i.e. anticipation, inclusion, reflection, and responsiveness.
The dimension of anticipation focuses on researchers investigating what is known, what is possible and what is likely in the field. This includes scenario building, making an assessment of their plausibility through interaction with experts as well as the general public, and the stimulation of an open and multidisciplinary collaboration. This we did through surveys, train debates, and through visiting conferences to learn about developments in the field and to make connections.
Subsequently, inclusion targets the process of open innovation and user-centered design. It focuses on transparency and collectively challenging regulations and standards. Grove-White et al. (2000) argue that the public conversation should stretch further to include the debate on future social worlds, while critically rethinking the ‘social constitutions’ inherent to the technological choices – that is, the ethical, political and social implications of the development. This we did during inclusion processes as the train debates and the expert discussion in Stirling.
Throughout the project, the process of reflectivity is continuously going on. We, as scientists, are used to professional self-reflexivity during the complete product development process. Our team continuously challenged our detection and we even had an inter-team detection method hacking challenge. However, as was stated by Wynne et al. in 1993, responsibility makes reflexivity also into a public matter. According to Stilgoe et al. (2013) reflexivity demands scientists to publically combine their scientific and moral responsibilities. This has been a prominent focus from the choice of our topic till our final design as can be seen from our interaction with the many stakeholders involved and the design requirements we derived from that.
Lastly, we responded to all stakeholder input by making a value sensitive design by which we managed to answer all needs and preferences of our stakeholders to come to an optimal method.

1. Anticipation

As a first stage in Responsible Research and Innovation we focussed on addressing the need for gene doping detection as well as on making an assessment of the challenges constituting gene doping with respect to future worlds.

1.1 Relevance of Gene Doping Detection

Gene doping caught our team’s interest at an early stage. Having often been confronted with the application of gene technology to humans and animals, we got fascinated by the concept of gene doping, its challenges and opportunities as well as its social relevance. Due to a lack of an implemented detection method it is hard to assess whether gene doping is currently happening. This is then of course always the first question people ask us. How relevant is your research? We can say it is a more eminent threat than you might have expected.

Let us start with the timeline in figure 1. Here some of the most prominent events in gene doping development are sorted in time and as it appears gene doping might already be happening.

Figure 1. Timeline of gene doping use and development in society.

On top of the articles supporting the suspicion and availability of gene doping, we wanted to assess the topic further, which we did through train debates and public surveys complemented by athlete interviews and contact with the Dutch Doping Authority as well as sports organisations as NOC-NSF, the Dutch National Sports Organisation, and a sports psychologist. From our surveys it became apparent that in The Netherlands 16% of the general public would like to use gene doping for performance enhancement without necessarily ascertaining its safety, in China this is 55%. These high figures amongst the general public together with the enormous pressure that is put on athletes give an indication of the need for detection. As a sports coach in Stirling pointed out, given the huge amounts of money going on in doping development compared to detection, gene doping will already be happening for sure.

Figure 2. Statistics on the willingness of the general public to use gene doping for performance enhancement in The Netherlands and The People’s Republic of China based on 181 and 126 respondents respectively.

Given the huge amounts of money going on in doping development, gene doping will already be happening for sure!
Sports Coach Stirling

1.2 Future Gene Doping Challenges

After we evaluated the relevance of gene doping detection, we focussed on the challenges of gene doping in the broadest context. We made a Stakeholder Map incorporating all stakeholders and their values. From this list that includes amongst others the doping authorities, athletes, sports clubs and coaches as well as physicians and fans, we identified our strengths and weaknesses. From there we grouped the challenges involved in gene doping in the following categories: health (both private and public, global and intergenerational), responsibility and social inequality. As it became apparent during the expert discussion in Stirling it are exactly these topics that make gene doping different from conventional types of doping.

These common values we addressed with the creation of our detection method and we discussed the topics in our expert discussion at the University of Stirling, where we talked about why gene doping detection is so important. Watch the movie on this discussion here.

2. Inclusions

In Anticipation we discovered the topic of gene doping in the broadest sense, both scientifically as well as ethically and socially. Subsequently, we took it further as a part of the inclusion process to involve as many people as possible for optimal design requirements for everyone. Here are some of the approaches we took in involving people from science, from sports as well as the general public.

2.1 Science

Hackaton

Figure 3. Advertising our Hackathon.

From our surveys we knew that 98% of the public feels strongly about maintaining strict doping controls. People feel that sports is only moderately fair and 75% is afraid of gene doping becoming a big problem in sports. Also, people generally feel they are not as up to date with developments in gene technology as they would like to be. These figures prompted us to involve the general public with a little programming experience in the fight against gene doping through the design of possible gene doping sequences.

On October 5th, 2018 we therefore organised a Hackathon at the Cyber Security Week in the Fokker Terminal in The Hague. The goal: engaging the public and especially computer scientists in developing their own gene doping sequences. We developed a software tool that learns from the ever growing database our participants helped create. This way, we improve gene doping detection together, so that we are able to detect new approaches in gene doping and to be one step ahead of the doping developers. We think that together we are stronger, inspiring each other. Many computer scientists joined our event and provided us with useful input from a different perspective.

Figure 4. The Hackathon at the Cyber Security Week in the Fokker Terminal in The Hague.

Stirling Expert Discussion on the Future of Gene Doping

We met professor Dimeo, Associate Professor in Sport from Stirling University, at the VVBN conference in Utrecht on May 17. After that inspiring meeting, we stayed in touch through a Skype call and email contact where we asked him questions on social aspects of doping as athlete privacy, behaviour, regulation and education. This resulted in mutual interest in each other’s research activities upon which we were invited to give a seminar on our project for experts in the field of doping and genetics at the University of Stirling on August 30th 2018. After this seminar we organised a discussion on how gene doping is different from currently more conventional types of doping and on how to best react to these differences, through regulation and/or education. This led to interesting discussions pointing at a general lack of education on gene doping and to the responsibility for germ line infections that cannot be transferred to second-generation athletes. The outcomes of the discussion are more elaborately described below in Outcomes and Implications. In the drop-down below the case studies we prepared for the discussion can be found.

Figure 5. Twitter comments on our seminar and expert discussion in Sterling, showing the impact and some new questions that arose.

Outcomes and Implications

The discussion took off very well, resulting in many opinions coming from different groups. The list of points that were identified to differ for gene doping compared to other kinds of doping is given in figure 5. The overall conclusion is that more attention should be paid to educating athletes on the risks above just regulating. Many athletes are not educated well about doping (and gene doping) and would therefore easily trust coaches etc. at the sports facility on their word to take in something of which they themselves are not able to oversee the consequences.

Figure 6. List of the differing factors between gene doping and more conventional doping.

Another topic adresses was that we cannot easily say that gene doping could become ‘safe’ at some point. Possibly it becomes apparent that we have been messing around with some feedback loops which has broader health implications on longer terms after some years. We cannot know with some initial studies. It is this what makes that we should be keep prohibiting gene doping according to the majority of experts present at the discussion. On top of this, differences in accessibility which you also already see in training facilities and equipment were used as arguments against gene doping.

Furthermore, it was brought up that there will always be differences between athletes, due to inherently different responses to gene therapies. Therefore, if everyone would be using gene doping, it is just like taking a step to somewhat higher performance, which will then level out again in time. So what would we achieve with doing it?

As became apparent in the discussion, the World Anti-Doping Agency (WADA) is not very open about gene doping to athletes and scientists. However, according to the experts at the conference, openness and involvement of the community could help a lot with the development of detection methods. This reinforces the community strength approach we take with for example the hackathon.

Engagement in Asia

The way people value sports is just as diverse as the people who love it, all around the globe. That is why it is important to weigh opinions not only in the Netherlands, but in the Peoples’ Republic of China as well. During our time there organising the iGEM Eurasian meetup, we spoke with Dr. Li Wei at NIFTY prenatal screening, who works with cell free DNA as well. He confirmed our assumptions on the cfDNA levels in the blood and outlined several possibilities of detecting it, discussing the advantages and disadvantages of next generation sequencing with us.

In addition, we spoke with Mr. Cao Jun, CEO of Sports Genomics Inc., on the future of genetic enhancement in amateur sports. His department’s main focus lies with helping people choose a sport that fits them based on their genetic information. For them, the border lies with reading the genetic information and recommending a course of action based on this, not enhancing.

2.2 General Public

Train Debates and the Public Opinion

On the June 26th, we extended the Belgian Biotechnology Day to The Netherlands. We wanted to open up the discussion on biotechnological subjects with a broad public. In order to find a diverse audience we organised train debates all over The Netherlands. The topic we chose was gene editing, which at the same time provided us with valuable information for our project. We spoke with people with radically different ideas and background. We even happened to talk to a professional soccer player who was, anonymously, quite open in admitting he would use gene doping if it was safe and undetectable.

If gene doping is safe and undetectable, then everyone would use it. So why not me? Anonymous Athlete

Figure 7: The paths we travelled by train throughout The Netherlands to engage in discussions with people.

Before we entered the trains we moreover had several hypotheses regarding the acceptation of gene technology in society. From our surveys we found that 16% of the public responding to our surveys in The Netherlands would like to use gene doping for performance enhancement. On top of this, 90% of the respondents indicate not to be afraid of gene technology. Moreover, 75% thinks gene doping will be a significant problem. Given the extra pressure on athletes to perform well, these statistics provide us with an alarming background to believe athletes will be using gene doping. All in all, the debates were extremely useful to have extra input as well as to educate people, giving them an insight in the newest gene technologies.

On top of this, we carried out surveys on gene technology and its acceptance in the streets, busses and metro’s in China. Interesting is the general acceptance of gene technology in both countries with around 90% not being afraid of gene technology. Privacy appeared not to be a big issue to people and 10 per cent of the respondents even indicated they would like to use gene doping for performance enhancement if this would invoke their death within 5 years. More information on the scientific background of the set-up of our surveys can be found on the Education and Public Outreach page.

2.3 Athlete and Sport Institution Interaction

Athletes

Apart from interaction with the general public and the diversity of experts present at the discussion in Stirling, we find it highly important to talk to athletes to see their perspective and take their experience and values into account.

Cameron Brodie

Cameron Brodie is a former professional swimmer, Scottish Record Holder (6x), Britisch Champion (2015) and Commonwealth Games Medalist (2x), who performed at this top level next to his studies at the University of Stirling. He has only had experience with urine tests, the preferable testing method. However, he did say that blood tests would not be a huge problem, since as an athlete being in the competition is worth that.

If blood testing necessary that is just the way it is, the testing is just inherent to sports and as an athlete all you want is to be in the competition so you have to comply. Cameron Brodie

According to Brodie, the pressure to perform well is “really tough”. He can imagine young athletes being vulnerable to people approaching them with gene doping opportunities. It was only in lectures of his sport related University study program that he first learned about gene doping. He himself, would not like to use it though, because one cannot oversee the consequences.

Moniek Nijhuis

Being rewarded for your hard work is valuable for sporters. Moniek Nijhuis, finalist Olympic Games 2012 and medalist at multiple European and World Championships, told us her story about one of the bronze medals she won at the European Championships 2013. Two years later, this bronze medal turned out to be worth silver due to doping usage by one of her opponents. However, her moment of euphoria on the stage, which is the moment that sporters are striving for, will never return.

I just wanted to know that what I achieved is purely due to my own power and efforts. That still makes me feel good and at peace. Moniek Nijhuis

Watch her view on doping use here.

Sports Organisations and Athlete Surveys

We contacted several sports organisations including the Court of Arbitration for Sport and a sports psychologist, Jef Brouwers. Both were not aware of any cases of Gene Doping. Mr. Brouwers did indicate however that he is aware of athletes carefully selecting their partners to have children that hopefully will perform well in sport again.

Apart from this we contacted the National Dutch sports organisation, the NOC-NSF, to hear about their experiences with gene doping. However, as they pointed out, they are not very familiar with the concept and the idea of it actually happening. They wanted to help to find out about the prevalence of gene doping though and send out our athlete survey to top level athletes. What became apparent from the athlete surveys is that athletes highly value quick detection with a result within a few days. Furthermore, athletes do not mind privacy invasive tests too much, since they see it as inherent to the desire to be in sports. This, to us, does not mean we should not take athlete comfort and wellbeing into consideration.

3. Reflection and Responsiveness

3.1 SWOT

As a result of our flourishing inclusion process, we had much valuable input from many different backgrounds. This prompted us to refine the analysis of our strenghts, weaknesses, opportunities and threats (SWOT) (Hill et al. 1997) to distill our core values from our challenges. Subsequently, we integrated all values and feedback into our design requirements and did we reflect on future implications and applications of our method based on our SWOT analysis.

Figure 7. An analysis of the strengths, weaknesses, opportunities and threats of ADOPE. Clicking on the terms will redirect you to the product design page where we make a further analysis of our product itself.

Based on the interaction with all stakeholders we then created a Value Sensitive Design to improve our strengths and reduce our weaknesses to satisfy everyone’s needs and preferences. In figure 8, an overview is given of our values, how they are related to values we identified through interaction and the design requirements that we implemented based on this. You can click on the terms to read the stakeholders that have influenced us in every step as described below.

Figure 8. Interaction figure of important stakeholder contact. Click on the terms and be directed.

3.2 VSD

3.3 Influencers

There have been many people that have had impact on our project. Here we list the ones that have directly impacted the paths we walked in our project.

Sample Preparation

The Dutch National Blood bank, Sanquin, has been of great influence for the development of our sample preparation. Sanquin is specialised in blood analysis and has it's own research departments to keep improving and developing new methods for the analysis of blood. Sanquin is responsible for all donor blood in the Netherlands, but is for example also specialised in tests focusing on the fetal cell free DNA in a mother's blood. This knowledge about analysis of DNA extracted from blood was exactly what was needed to develop a secure and optimized sample preparation for our project, ADOPE.

Two visits were made to Sanquin, one general introduction visit and one specialized visit where the specific DNA extraction method was taught to some of us. Aicha Ait Soussand and Ellen van der Schoot of the Experimental Immunohematology group of Sanquin helped us by explaining how they work with small fragments and DNA extraction and gave us access to their optimized extraction protocol used with the QIAmp DNA Blood Mini Kit. Since isolation of fragmented cell free DNA out of blood and white bloodcells can be quite a hard challenge because of the low concentrations, the experience of Sanquin helped a lot in optimizing our DNA extraction method. In addition, they pointed at the delay in red blood cell development, which gave us the idea to extend our model to include the whole process of gene doping and its effect.

Targeted Sequencing

Professor on Therapeutic Gene Modulation Hidde Haisma gave us insight in the most attractive methods for athletes for gene doping. Also, he gave us information on the detection possibilities for gene doping, for example the presence of exon-exon junctions due to the removal of introns, a distinct promotor for increased expression and viral vectors to penetrate into the human cells. Furthermore, he inspired us with his research in whole genome sequencing for gene doping detection and his limitations concerning data analysis. Reducing our data output for less complicated data analysis became one of our requirements for our gene doping detection method.

I expect athletes that would be using gene doping now to use either plasmids or adenoviruses as vectors. Prof. Hidde Haisma, Gene Doping Detection Expert

Alina Ham, Gerard Coyne and Angelica Vittori from Oxford Nanopore Technologies inspired us to adapt our initial idea, which would involve detection of target sequences based on dCas9-affinity and subsequent nanopore blocking. The forces exerted by the motorprotein were suggested to overcome dCas9 affinity, and were most likely to push off the DNA-binding protein. This important advice made us change our project from a detection method based on signal absence towards a methodology striving for targeted sequencing.

Fusion protein for targeted sequencing and library preparation

The idea for our fusion protein came through several phases. We read about Zinc finger and Transcription activator-like effector nucleases (TALENs), but wanted to improve on the versatility to anticipate the plethora of changes that could be made to the genes used as gene doping. Therefore, we came up with a Cas9 based protein with a flexible guide RNA library after elaborate discussions with amongst others prof. Stan Brouns. Later, during a presentation at the Delft Health Initiative, CRISPR experts challenged our approach because of the on and off target effects of dxCas9 , but praised our idea for its versatility and thereby its probably functionality.

Prof. Chirlmin Joo and Viktoria Globyte advised us on this functionality of our fusion protein in its early stages, providing us with a confident start of the wet lab fusion protein production.

Cas9 scanning and kicking off from non-target DNA is faster than transposase cutting. Professor Chirlmin Joo, Director of the Kavli Institute of Nanoscience

Multiplexing and Barcoding

Professor Hagan Bayley from Oxford University, one of the founders of Oxford Nanopore Technologies, pointed at the enrichment of our sample. This prompted us to focus on an extensive sample preparation. On top of this, prof. Bayley said that multiplexing and accompanying barcoding would be a big advantage, which we then set out to implement, improving upon an existing iGEM barcoding tool.

ADOPE’s ingenious approach to foreign gene detection pushes the frontiers of forensic analysis. Professor Hagan Bayley, Oxford University, Cofounder of Oxford Nanopore Technologies

Prescreen

Olivier de Hon, principal scientist at the Dutch Doping Authority, gave us highly valuable insights into the requirements that the doping authorities set for a detection method. A conversation with him resulted in our focus on the nanoparticle based prescreening method.

The initial costs are not very important in doping detection development. What does matter is that we should be able to efficiently upscale the detection. Olivier de Hon, Dutch Doping Authority

Minimizing out of competition testing

As became apparent from the interviews with Moniek Nijhuis and Cameron Brodie, out of competition testing can be highly privacy invasive in the sense that athletes always need to keep track of where they go. Therefore, we found it important to assess how to reduce testing time and optimally schedule possible testing points to have least impact on the athletes every-day life. To determine the optimal detection point for gene doping, we developed a model of the human body response to EPO gene doping, incorporating the blood cell development in the bone marrow based a suggestion by the Dutch National Bloodbank Sanquin.

Furthermore, the initial Skype call with Prof. Paul Dimeo from Stirling University prompted us to focus on athlete behaviour, to think with them and thereby be a step ahead. We focussed on different administration methods for gene doping (intravenous and intramuscular) and on the effects of microdosing EPO gene doping. In this way we determined that our method could best be included in out of competition testing See our model.

Safety-by-Design

For our team safety does not only come first. We prefer to say “safety always”. That is why we actively incorporated safety throughout our project, from the topic choice, focussing on responsible use of synthetic biology, to the product development, consisting of a cell free device. The RIVM, the Dutch National Institute for Public Health and the Environment, advised us on this. We concluded that from an environmental perspective unregulated gene doping use throughout society might pose another threat and thereby a reason for detection, at least in sports, and further awareness throughout society. Please click on the image below to get a better overview of how we incorporated safety in our project.

The vectors people in society would use for gene doping might in time also pose an environmental threat, depending on the vectors. Cécile van der Vlugt

The Future of our Method

The Dutch Doping Authority has shown interest in the implementation of our method. More on this can be read on the Entrepreneurship page. On top of this, we identified another market interested in our gene doping detection method: horse racing. Gene doping lately receives huge interest in the horse racing world. Earlier this year, the 37th Asian Racing Conference in Seoul specifically focussed on gene doping (Bloodhorse, 17 May 2018), illustrating the imminent threat of gene doping in this world.

The race to produce the first genetically engineered super-horse has already begun. Argentinian scientists have started performing gene editing on horses, and it is speculated that the first super-horse is likely to be produced by them as early as 2019. The gene that they are targeting is myostatin, which is crucial for muscle development. In other words, it could allow a horse to jump higher and run faster. Dr. Teruaki Tozaki, technical advisor for the Laboratory of Racing Chemistry in Japan

This year the University of Pennsylvania School of Veterinary Medicine even received $300.000 dollar towards the development of a gene doping detection system according to Kim Yuhl in the Online Pennsylvania Play Magazine (Yuhl et al. June 22, 2018). The Dutch Horse Racing Association has shown great interest in our product as we discuss on our Entrepreneurship page.

Apart from direct gene doping applications, the Dutch National Bloodbank Sanquin is working on methods for prenatal screening of diseases for which they have shown great interest in our method. This is only one of the variety of extended applications of our targeted sequencing method. The Stan Brouns Lab at Delft University of Technology is so enthusiastic about our project that they want to develop our method of targeted sequencing for broader applications. Nevertheless, the fight against gene doping might be continued as well depending on a big grant application we started. Read more on the steps we have been taking towards the future applications on our Entrepreneurship page.