Overview

We want sport competitions to be fair and athletes to be protected against gene doping, the misuse of gene therapy in sports. People caught up in the rat race of doping development underestimate the implications of gene doping, that can stretch beyond sports into public health and social inequality. To get an overview of the design requirements for gene doping detection, we organized a discussion with experts, athletes and coaches at the University of Stirling. We implemented athletes’ wishes regarding invasivity, privacy and testing frequency into our detection method. Further interaction with stakeholders such as the Dutch Doping Authority and Oxford Nanopore Technologies made us add a prescreen and a barcoding tool to our detection method for versatile, high throughput and reliable detection. As a final challenge, we invited engineers to hack our detection method, and used their collective strength to improve our algorithm and anticipate future gene doping developments.

Conclusions Approach

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 the future.

1.1 Relevance of Gene Doping Detection

Due to a lack of an implemented detection method it is hard to assess whether gene doping is currently happening. We can say now however that it is a more eminent threat than you might have expected.

Gene doping is a real problem. As the science improves the usage will expand. Testing is going to have an impact, in lots of diverse ways. This group have expertise and are willing to engage in transparent, open debate. I think @wada_ama and many others should talk to them. https://t.co/zrO6GQFSVz

— Paul Dimeo (@pauldimeo2) August 30, 2018

In the timeline in figure 1 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.

Timeline

We assessed the topic further through train debates and public surveys complemented by athlete interviews and contact with the Dutch Doping Authority as well as sports organizations as NOC*NSF, the Dutch National Sports Organization, and a sports psychologist. We found that up to 55% of the general public would like to use gene doping for performance enhancement without necessarily ascertaining its safety. 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.

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. More on the surveys in The Netherlands and China can be found on the Education and Public Outreach Page.

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 grouped the challenges involved in gene doping in the following categories: health (both private and public, global and intergenerational), responsibility and social inequality. As became apparent during the expert discussion in Stirling, exactly these topics make gene doping different from conventional types of doping.

Health Responsibility Social inequality

We addressed the challenges described above with the creation of our detection method and discussed the topics in our expert discussion at the University of Stirling. Here we talked about why gene doping detection is so important and why it is extremely important to unite strengths. You can watch the movie on this discussion below.

Video 1. Our expert discussion on the future of gene doping.

2. Inclusion

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

Here we elaborate on science related events that have largely influenced our project. At reflection and responsiveness we elaborate on individual stakeholders that have changed our project.

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. These figures, and the strong collective spirit that was stressed in Stirling, prompted us to involve cyber security specialists in the fight against gene doping through the design of possible gene doping sequences.

On October 5th, 2018 we therefore organized 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. In 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 3. An impression of our Hackathon where we challenged cyber security specialists to hack our method.

Stirling Expert Discussion on the Future of Gene Doping

Very interesting presentation. Really impressed about the openness of @TUDelft_iGEM in presenting a model for testing #genedoping. Definitely a turning point to something new on this topic... @wada_ama

— Nicola Busca (@Bogpolis) August 31, 2018

Figure 4. Twitter comments on our seminar and expert discussion in Stirling, showing the impact and some new questions that arose.

Case 1: Intergenerational Responsibility Case 2: Where do we take it? Outcomes and Implications

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 organizing 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.

During our time in China we furthermore handed out surveys in the streets, buses and subways. This gave some very interesting results as is further described on our Education and Public Outreach Page.

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 synthetic biology with a broad public. In order to find a diverse audience we organized 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.

Figure 6. The paths we travelled by train throughout The Netherlands to engage in discussions with people.

More information on the scientific background of the set-up of our surveys and the results we achieved in both The Netherlands and China (during our visit to China for the EurAsian Meetup) can be found on the Education and Engagement 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), British 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.

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

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.

Watch her view on doping use here.

Video 2. An interview with Moniek Nijhuis, professional swimmer.

Sports Organizations and Athlete Surveys

We contacted several sports organizations 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 organization, 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 set the initial steps to send out our athlete survey to all Dutch top level athletes. What became apparent from the athlete surveys we had already send out is that athletes highly value quick detection with a result within a few days. Furthermore, athletes tend to not mind privacy invasive tests, since they see it as inherent to the desire to be in sports. To us nevertheless, athletes' comfort and well-being remain top priority at all times.

3. Reflection and Responsiveness

Apart from the science related events, we have talked to many individual stakeholders and integrated their feedback into our design, as can be seen from our Value Sensitive Design.

3.1 Value Sensitive Design

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. Below you can read more about how the stakeholders have influenced us in every step of our project.

Figure 8. Value Sensitive Design flowing from the core values of ADOPE through stakeholder values to the implemented design requirements.

3.2 Influencers

There have been many people that have had impact on our project as can be seen in figure 9. Below we list the ones that have directly impacted the paths we walked in our project and how these stakeholders influenced us.

Figure 9. Interaction figure of important stakeholder contact.

Sample Preparation

The Dutch National blood bank, Sanquin, has been of great influence for the development of our sample preparation. Sanquin is specialized 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 specialized 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 blood cells 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.

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 motor protein 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.

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.

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.

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 blood bank Sanquin.

Furthermore, Prof. Paul Dimeo from Stirling University prompted us to focus on athlete behavior, 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. In the image below an overview is given of how we incorporated safety throughout our project.

Figure 10. Infographic Safety-by-Design.

References Infographic

The Future of our Method

The Dutch Doping Authority has shown interest in the implementation of our method and the Delft Sport Engineering Institute is interested in further support of our research. 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.

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. On top of this, the RIKILT, the Dutch Research Department for Food Safety at Wageningen University and Research, world’s number 1 university for food technology, has shown interest in our method for safe food applications. These are only a few 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.

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