Safety

Introduction

For a general overview we identified possible risks in our lab and in synthetic biology in general. Firstly we concentrated on general lab safety.

General wet laboratory safety:

We worked in a BSL-1 laboratory, so the general safety measurements for such a facility were kept upright at any given time point. It was absolutely mandatory to wear protective clothing (labcoat, gloves, and closed shoes). Coordinated and thoroughly work in the laboratory is essential for establishing and maintaining safety standards in our wet lab with minimized risks for everybody working there. Besides receiving the mandatory safety training (q.v. safety form) we divided the laboratory in work areas for different parts of our project/ stepsin the laboratory work.

Besides the distribution in the main laboratory where cloning and gel electrophoresis took placea large part of our wet lab was located in the cell culture. Working sterile under cell culture fume hood benefitted the safe handling of our system and demagnified the risk of parts escaping the laboratory. To further prevent contamination of the environment we autoclaved all our S-1 contaminated waste. This altogether ensured a safe environment to work in.

Viral vector safety

1: Are we creating amphotropic MLV derived retroviruses during our research?

No, we are creating ecotropic replication-deficient MLV derived retroviruses. In contrast to amphotropic viruses these are not able to infect human cells under normal conditions. Explanation

In order to produce our viral vector we are inserting two different plasmids into a human packaging cell line (Phoenix). This cell line is carrying the gag, pol and env genes, which are needed for the synthesis of the virion. When transfected with a s called transfer plasmid these cells are producing replication-deficient MLV viruses. The virus can be harvested in the cellculture supernatant afterwards. The env gene is responsible for the ecotropic nature of our viral vectorcapsid. The ecotropic MLV is highly specific for the mCAT1 receptor only found on rodent cells (mouse and rat). (picture control vs HEK). Cells that were transduced are not able to generate viral particles because they are lacking the required genes for the production of the virion, even after stable integration. The work with ecotropic MLV is performed under BSL1 requirements.

We tested the specificity with different human cell lines to validate that normally human cells cannot be infected by our viral vector.

2. Is it possible to infect human cells with these viral vectors under experimental conditions?

Yes we infected HEK293T (Human Embryonic Kidney) cells with our ecotropic MLV.

Explanation

In order to infect non-rodent cells we decided not to pseudotype the virus itself but the, to be infected cells. This allows us to create a safe and controlled environment that allowed us to only transfect desired cell populations. Manipulating the capsid itself would have led to an increased need of safety. Because we wanted to distribute our viral vector throughout the iGEM community design it as safe as possible was our major goal.  Therefore we  transfected cells with the mCAT1 to specify them as targets for viral transduction. This allowed us to build safe BSL1 environment for everyone to express their genes to create a wonderful new world of transgene cells. 

3. How did we measure the safety of our viral vector in detail?

During our research we altercated with existing laws and created a catalogue of characteristics describing the safety of our viral vector. As a basis of valuation we studied the law of prevention and fighting of infection disease (IfSG) as well as the genetechnology safety edict (GenTSV).

Following the description of the law the criteria for human pathogenic genetechnically engineered organisms are: Transferability, infective dose, host range, possibility of survival outside of human host, the presence of vectors and means of dissemination, biological stability, allergenicity and toxicity.

3.1. Transferabiity, infective dose and host range:

Rodents are the target of our MLV, there are no cases described in which non rodent cells were infected by an ecotropic MLV. MLVs are transferred by fluids Non-rodent cells carry different glycosylation patterns at their CAT1 receptor and can therefore not targeted by the ecotropic MLV. The immune system of non-rodent mammalians provides different other barriers preventing an infection. The complement system of these mammalians is recognizing the MLV and is inactivating it. Furthermore a number of restriction factors are prohibiting an infection. Possible is a receptor blockage, avoidance of replication after penetration (Trim5alpha) or impede activation of retroviral elements in the genome (deaminases, Zink-Finger-proteins, micro-RNA, siRNA).

Our retroviral vectors are not air transmittable and are unstable under different conditions. For transduction a direct contact between virus and cell is mandatory. The cells need to be in the mitotic phase in order for the virus to integrate into the host genome. Human skin cells which are in the mitotic phase are located in the basal lamina. Above those is a layer of non mitotic cells protecting the skin from infections by viruses. Only with damaged upper layers a hypothetical infection would be possible. Besides the fact that cells without the mCAT receptor are tranduced at all this shows how unlikely a accidental infection with a retroviral vector in general would be.

We tested the half life of our viral vector at 37 degree celcius. (picture of half life). This temperature instability states another safety aspect. Accidental escaped viral vectors would be inactivated very rapidly. To prevent such an event the particles can be degraded by different methods. For example Chloroform, phenol, bleach, 70% ethanol, UV-light and low pH-values under 6.5. The pH-value of human skin is 5.5 therefore viral particles on the human skin are inactivated by contact. (picture of UV chemicals)

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