Safety

Introduction

iGEM teams all around the world are trying to find solutions for issues that might influence not only laboratory work routine but also daily life in the near future. Due to this fact it is very important to think about possible dangers arising from synthetic biology. During our human practice we experienced a strong desire of the public for solutions that not only have biggest possible efficiency but in first line are safe. First we encountered this attitude in the broad public while we tried to explain them synthetic biology in general (balloon action and mindmap). They were mostly concerned about possible safety leaks. After this experience we decided to take a look at what the experts of ethics and law are thinking about synthetic biology and therefore we attended the symposium: „Das Missbrauchsrisiko in den Biowissenschaften-Biosicherheitsrelevante Forschung zwischen Freiheit, Fortschritt und Verantwortung“ (Risk of misuse in biology- biosafety relevant research between freedom, progress and responsibility /Freiburg 2014/06/03). Here too most of the speakers were concerned about safety and security issues regarding this relatively new field of research.

For this reason we wanted to start LinkIT – gaining acceptances by overcoming fears. It is our strong believe that better education of the public alongside with identifying, occupying and minimizing the possible risks of our own project would benefit the general attitude towards synthetic biology.

The linking of safety and security is, in our opinion not only possible but mandatory. With people thinking that synthetic biologist around the world are creating Frankenstein it is almost impossible to build a trusted platform of sound debate. The spirit of iGEM is to perform projects that may be applicable in daily life. It is utopic to think that this will become real if we are not starting now to involve and educate the public. The shortcoming of such practices can be seen in modern gene technologies were a lot of applications might be possible and beneficial but the backing of the public is, especially in Europe not existent. This will also be limitations for research and application. You can have best ideas and lab-safety but won’t be able to bring them to the market because of lacking support and fear from the public.

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

	Environment	Public/Humans
Research and production	Safety risk by unintentional release from laboratory	Safe Laboratory work
Application	Danger of uncontrolled dispersal	Danger for health especially for medical therapeutically application

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, closed shoes). Furthermore we divided the lab-area in distinct working places, with spaces for cloning, gel-electrophoresis and western-blot.

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 distributed the laboratory in work areas for different parts of our project/ steps in the laboratory work. Besides the distribution in the main laboratory where cloning and gel electrophoresis took place a large part of our wet lab was located in the cell culture. Working sterile under extractor hoods 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 after separating it from the general waste.

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)

References

Miller DG, Edwards RH, Miller AD (1994) Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus. PNAS USA 91:78-82
Battini JL, Rodrigues P, Müller R, Danos O, Heard JM (1996) Receptor-binding properties of a purified fragment of the 4070A amphotropic Murine Leukemia Virus envelope glycoprotein. JVirol. 70 (7): 4387-4393
Stellungnahme der ZKBS zur Risikobewertung ecotroper C-Typ Retroviren der Maus, (Az.6790-10-41, April 1996)
Cornetta K, Moen RC, Culver K, Morgan RA, McLachlin JR, Sturm S, Selegue J, London W, Blaese M and Anderson WF (1990) Amphotropic murine leukemia retrovirus is not an acute pathogen for primates. Hum. Gene Ther. 1 (1): 15-30
Rother RP, Squinto SP, Mason JM and Rollins SA (1995) Protection of retroviral vector particles in human blood through complement inhibition. Hum Gene Ther 6: 429-235
Pansiero MN, Wysocki CA, Nader K, Kikuchi GE (1996) Development of amphotropic murine retrovirus vectors resistant to inactivation by human serum. Hum Gene Ther 7 (9): 1095-1101
Fields Virology 5th Edition (2007) Lippincott Williams & Wilkins
RetroMax-System-Instruction Manual-IMGENEX
Naviaux RK, Costanzi E, Haas M, Verma IM (1996) The pCL Vector System: rapid production of helper-free, high titer, recombinant retroviruses. JVirol. 70 (8): 5701-5705
The Journal of Gene Medicine Clinical Trial site http://www.wiley.com/legacy/wileychi/genmed/clinical/8
Fehse B (2007) Insertionsmutagenese – Implikationen und Möglichkeiten der Vermeidung. Schwerpunktprogramm 1230 der DFG
Stellungnahme der ZKBS zu häufig durchgeführten gentechnischen Arbeiten mit den zugrunde liegenden Kriterien der Vergleichbarkeit: Gentransfer mit Hilfe retroviraler Vektoren(Az. 6790-10-41, Oktober 2007)
Methodensammlung der LAG (2009) Quantitativer Nachweis von Lentiviren (HIV1)-RNA mittels Real time RT-PCR.
Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (April 2009) Ringversuch „Quantitativer Nachweis von Lentiviren (HIV1) RNA“-Ergebnisbericht
Levy, JA (1995) The Retroviridae. Plenum Press, NY
Hacein-Bey-Abina, S et al. (2002) Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. New Engl J Med 346 (16): 1186-1193.
Hacein-Bey-Abina, S et al. (2010) Efficacy of gene therapy for X-linked severe combined immunodeficiency. New Engl J Med 363 (4): 355-364.

Symposium on the 3^rd of July

As our project is dealing with viral vectors, we care a lot about the issues of biosafety. Among other things we took part in an interdisciplinary symposium dealing with “The risk of abuse in biosciences”. We heard presentations about the biosafety-relevant research in between freedom, progress and responsibility. There were two to three experts/ speakers for every of the following topics:

scientific basics of biosafety-relevant research
biosafety in practice including research codes and research funding
legal framework of biosafety-relevant research in international perspective
risk ethic, research freedom and responsibility from a philosophical and sociological point of view
biosafety relevant research and security against B-weapons

After every topic we had time for questions and discussions. In a short summary it is to say that research is very important, however there is always a risk of abuse. But this depends entirely on the person working with the subject of interest. In addition, there is also a special treatment for working with DURCs (dual use research of concern). Moreover, you have to be careful publicating your research results, but there should be no restrictions for publications.

The symposium was organized by an institute of public law and unfortunately many of the participants were not familiar with a lot of biological background knowledge. So speaking about these themes fuelled baseless fears and a lot of skepticism. As a conclusion we remembered that people needed to be more sophisiticated about the huge and awesome profits of the synthetic biology. For that reason we organized the air balloon event and other policy and practice stuff.

Safety-Sheet regarding the work with viral vectors

To expand the usual safety- and check-in-forms we developed an additional safety-sheet which not only increases safety for scientists working directly with viral vectors but also aims to provide an easy to understand tool for people not involved in laboratory work. This is also part of our goals we want to achieve with LINK-it, to increase the acceptance of the public by giving them greater insights.

We are providing the pMIG (viral vector) as a tool for easy gene delivery and generating cell lines under BSL1 conditions to the iGEM community. This spreadsheet, as a checklist for safety standards supplies future teams with the possibility to fast and on the point inspection of their projects safety (LINK registry).

General safety of the viral vector

Criteria	Explanation	Our Project	Check
Human pathogenesis	Is it possible that the viral vector causes any illness or irritation in humans	We tested our viral vector for potential infection of human cells. Therefore we tried to infect HEK293T (human embryonic kidney) and A549 (lung cancer) cells. Non of our results indicated any infection of those cells. (LINK)	✔
	Viral vectors differ from natural viruses. A virus needs at least three different genes to replicate. These are the gag, pol and env genes. In order to ensure higher safety a lot of viral vectors lack these genes. The viral vectors themselves can not reproduces themselves. The vector can integrate itself into the hosts genome, a process coined transduction, but cannot create new viral particles. Its more or less a cul-de-sac for the viral vector.	We sequence our viral vector to ensure the lack of the three viral genes (gag, pol, env). In order to generate the viral vector we used the Phoenix cell line which harbors the three genes under different non-viral promoters to minimize the risk of recombination. (LINK registry seite)	✔
Transmissibility	How can the viral vector be transferred between cells/organisms. There are different means by which pathogens can be transmitted: by air, (body) fluids or by contact.	We performed a extensive literature research on our viral vector prior to our lab work. We found that the viral vector we are using is solely transmittable via fluids. (LINK text) Nevertheless we performed all steps involving the viral vector with maximum carefulness. (LINK safety lab)	✔
Hostrange	Cells from which animals can be infected by the viral vector.	The MMLV used in our project is highly specific for the mCAT1 receptor. This receptor variation of the CAT1 receptor is only found in rodent cells (mouse, rat. Human cells also carry a CAT1 receptor but with a different glycosylation motive and are therefore not recognized by the viral vector. ). (LINK receptor)	✔
Survival outside of host	How long is the timerange of survival of the viral vector until it becomes non-infectious.	Besides literature research we wanted to test our viral vector for its half-life time. We started at a given time point and measured the exact percentage of infected murine cells. We repeated this for several time points using the same viral vector which was stored at 37 °C. Our findings of a half-life of ~6 h matched those found in literature. (LINK literature and wiki )	✔
Presence of transmitter	Is it possible for natural transmitter to come in contact with the viral vector.	The only natural transmitter of our viral vector are rodents. Besides not having any rodents in our lab, the vector itself with its non-replicability ensured the safety standard.	✔
Usage	Is the vector used in only under lab conditions for research or is it going to be used in humans for clinical applications.	The MMLV is, in our project only used for research purposes. The viral vector can cause leukemia. In clinical applications using viral vectors the amount of used vector is considerably higher. Furthermore the specificity viral vectors must be altered to enable the infection of human cells. With our significant lower virus titer and lower used amounts of viral vector suspension transduction is more than unlikely. (LINK zu links des textes)	✔
Additional safety measurements	Every lab that works with genetically modified organisms (GMO) must ensure a certain standard of safety (Germany: Anhang I.2 GenTSV).	After careful research and consultation of our administrative department safety officers the viral vector used in our project falls under the BSL1 category. To ensure safety at BSL1 we received safety training. (LINK zu safety form, BSL1 guidlines)	✔

Team:Freiburg/Content/HumanPracticeAndSafety/Safety

From 2014.igem.org