In the Netherlands, the RIVM (the Dutch National Institute for Public Health and the Environment) conducts research to make sure that we keep ourselves and our environment healthy. This also includes opening discussions about the developments in synthetic biology and the safety issues regarding this. More information about the general safety aspects of our project can be found here.
This year the RIVM encouraged the Dutch iGEM teams to 'think before doing': think about how we are going to incorporate safe-by-design in our project.
Safe by design
Below you can find a short description of our implementation of "Safe by Design". If you want to know more about certain topics use the arrows.
What is Safe by Design
Safe by design is a way to incorporate safety in every design aspect: start to think about safety issues and risks even in the design stage of the project to identify the risks beforehand instead of considering them afterwards.
The first step to incorporating safe-by-design is to think about what is meant by safe-by-design. Safe-by-design can be seen as a way to incorporate safety in every design aspect. By starting to think about safety issues and risks even in the design stage of the project, risks can be identified beforehand, instead of considered afterwards. Key to this is to not only to think about the components (DNA, chemicals, proteins) which are used, but also about the outcome of the reactions taking place. For instance, if a chemical reaction could produce unwanted by-products, these should also be considered.
Our project
In our project we do not design a bacterial system, but we work with proteins which can form a network. The scaffold protein and its binding partner are well studied at our university. The small molecule inducer is a toxin, but research has shown that it has no measurable negative effects in high concentration. The proteins will be used in our project to make a network, we have modeled this network formation to provide us with more information about this.
For our project we use Escherichia coli as a host organism, which is an organism of risk group 1. This organism is then transformed to express our proteins constructs: a scaffold protein (14-3-3) and its binding partner (CT52). Since Escherichia coli is only used to produce the wanted proteins, we are not developing a bacterial system in which safety mechanisms like a kill switch can be induced. However, we can look at the safety of our proteins and at the safety of the network that is formed.
First of all, 14-3-3 and its binding partner are well studied proteins at our university: the sequence is known and our lab has experience with the expressed protein and optimized its amino-acid sequence for different applications. This eliminates risks present if one would start working with ‘new’ or relatively unknown proteins. For network formation, we will use a small molecule inducer, fusicoccin. Fusicoccin is a toxin produced by fungus and has detrimental effects on plants. However, research has shown that fusicoccin in a high concentration does not show measurable negative effects on mammalian cell lines, so it would be safe to use as an inducer.
Besides the proteins itself, we also thought about the network formation. Since the interaction between 14-3-3 and its binding partner are well known, we have worked on creating a model. This way it is easy to screen concentrations and conditions under which the network is formed, providing us with more information.
Application
The application of our project lies in encapsulating cancer cells. The idea is that an inhibition site is present which prevents the two proteins to interact. However, a cleavage site is introduced which can only be cleaved by proteases excreted by tumor cells, thus making it a specific process.
For the eventual application of our network we thought of encapsulating of tumor cells. More information on this application and the viability can be found here. The general idea is that the 14-3-3 construct contains an inhibition site which prevents network formation. This inhibition site is linked to the construct with a linker which contains a specific protease cleavage site. Proteases excreted by tumor cells can cleave this site, eliminating inhibition after which the network is formed. Since the cleavage site is specific for the proteases excreted by tumor cells, the network formation is thought to be localized around cancer cells.
What if it goes wrong
To prevent unwanted gelation, an extra cleavage site can be introduced, specific for another protease. When the network formation occurs at an unwanted location, this other protease can be added and the network will fall apart.
Rules & Regulations
General
Since our project involves the use of GMOs (Genetically Modified Organisms) we decided to take a deeper look into the GMO policy in the Netherlands.
First of all, the laws on GMOs in Europe are set in the European Biosafety Regulations,valid for all member states. These laws are valid for the contained use of genetically modified micro-organisms. In the Netherlands, Bureau GGO is the bureau responsible for providing authorization for activities involving GMOs. Bureau GGO is part of the RIVM and is commissioned by the Ministry of Infrastructure and the Environment.
When applying for a GMO permit, a risk assessment must be conducted. All properties of the GMO have to be taken into account as well as the activities involved with this GMO. This risk assessment eventually indicates the containment level at which the activities should take place. Five different containment levels exist: I, II-k, II-v, III & IV. These 5 levels are for the contained use of GMOs, when one wants to bring a GMO into the environment, an IM license is needed. (IM stands for Introduction into the Environment).
Next to Bureau GGO, COGEM is also involved in the GMO permits. COGEM stands for The Netherlands Commission on Genetic Modification and is an independent scientific advisory committee composed of scientists. After the risk assessment sent to Bureau GGO has been checked, COGEM will give an advice about whether or not the permit will be granted, based on scientific literature and expert judgement of the members.
Product Safety
Next to GMO policy, we also wanted to investigate the rules and regulations around the application of our project: the encapsulation of tumor cells. Via the RIVM we got into contact with Claudette de Vries, she works for the RIVM at the Centre for Health Protection. This centre focuses on the health impacts and risks to people of chemical and biological agents, with a focus to medicines among other things.
When looking at medicines, several medical product types exist: medicinal product, advanced therapy medicinal product (ATMP), medical device and in vitro diagnostic medical device. So roughly there are two categories: medicinal products and medical devices. When looking at the definitions from the directives 2001/83/EC and MEDDEV 2.
A medicinal product is a substance that exerts pharmacological, immunological or metabolic action or a substance that is administered with the aim to make a medical diagnosis. A medical device does not achieve its function by pharmacological, immunological or metabolic action. The function of a medical device is achieved by physical means: mechanical action or creating a physical barrier. Since our system can be used to encapsulate tumors, it can be seen as a physical barrier and thus as a medical device. For medical devices, the legal framework can be found here.
Next to the legal framework of the device itself, it is also important to verify the clinical safety of the device. In Europe, this is verified by a clinical evaluation. Manufacturers planning to sell a medical device in Europe are obligated to write a Clinical Evaluation Report (CER). In this report, clinical data is presented. This data may include clinical data from literature, clinical experience, or clinical trials. The clinical evaluation in the CER should comply with MEDDEV 2.7.1 revision 4,a guideline on medical devices. In the end, a CER should show that the device achieves its purpose without exposing users and patients to risk.
