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Definition of „artificial cell“ and „living machine“ in synthetic biology

 

Synthetic biology is still a young science field, which combines areas of engineering, chemistry, biotechnology, computer science and molecular biology.

The synthetic biology can be defined by their objectives. This definition encompasses four points. One goal is to gain new scientific knowledge about the processes within cells by trying to build cells from the bottom up or the top down. Another goal is to open up new fields of possible applications for synthetic modified organisms for example in medicine.  A key issue involves the creation of novel, non-naturally occurring organisms and structures, as well as modularization and standardization of repeatedly compassable components: Biobricks.  (TESSY).

 

Before discussing the definitions of „artificial cell“ and „living machine“ it is crucial to define to what extend can ever be spoken about a creation of life in synthetic biology.

The currently pursued research approaches in the synthetic biology do not aspire the creation of life "de novo". “De novo” can be explained from the Latin term “newly create, invent”(Pons). In connection with synthetic biology this implies the creation of new organisms without any foundation in already existing organisms. Up to this day researchers are working with organisms found in nature.

However, in the future scientist attempt to design and manufacture new biological systems, with no origin in nature. (Boldt et al. 2009). Creating new life is consequently one main goal of synthetic biology.

There are three different approaches aiming at the goal: the DNA-based device construction, genome-driven cell engineering and protocell creation (O´Malley et al. 2008).

 

The DNA-based device construction approach engages with the method of modularization, standardization and abstraction of biological constructs (Arkin 2008). Building a collection of biobricks and inserting them in organisms gives these new functions. Creating new, not in nature occurring species (European Commission 2003).

Model for this is the technical field. Genetic sequences are intended to function like a construction kit, working in every desired organism. For the following discussion, the sematic analogy between the genetic constructs and man made machines is further investigated. Apparent are the used terms „bioengineering product“ or „living machines“ (Deplazes ans Huppenbauer 2009; Tucker/Zilinskas 2006).

 

The genome-driven cell engineering approach aims to create a so-called „minimal cell“. Parts of the cell should be reduced to a minimal extend, leaving only necessary properties for survival. Minimizing the genome after the top-down-approach pursues the creation of an organisms as a „cassis“. A variable frame where every biobrick can be introduced into the genome. (Gibson et al. 2008)

 

The protocell creation approach seeks to build a new organisms „de novo“ after the „bottom-up-approach”(Luisi et al. 2006). Unlike the two other approaches the protocell-creation is not guided by an archetype  (Boldt 2008).

 

These three approaches raise different ethical questions.

The ethical implications using the appellation of „living machines“ and „artificial cells“ will be elucidated. Focusing on the implicated hybrid-character of such designation, with regards of the relation between machines and living organisms and the deceptive idea of an “artificial cell”. Also  the acquaintance arising from the determination between “artificial cell/living machine” is illuminated.

 

The term Der Begriff „living machine“ - a hybrid between machine and living organisms

Machine originates from the Latin term „machina“ and means „tool, artificial equipment“ and is defined as „a piece of equipment with moving parts that is designed to do a particular job.[..]“ (Pons; Oxford dictionary 1884).

It follows that machines are designed for a particular purpose, according to the plans of humans and adopt a predetermined, defined and by itself immutable function. Machines are per definition not in the position to plan and conduct their behavior autonomically. They cannot change actions by themselves and can only operate in a certain way with the environment. Crucial characteristic for a machine is the autonomy but not the material, organic or inorganic (Deplazes 2011).

 

In contrast to the heteronomous machine stands life itself. The discussion about the concept of life in synthetic biology is a complex and wide-ranging field, where the difficulties of a definition lie just in the diversity of life. It is noteworthy that the researchers themselves are trying to find a suitable explanation, as it is an objective of synthetic biology to create new life (Deplazes / Huppenbauer 2009). Here is a brief description how researchers are trying to define life or alive.

 

After a minimal definition each organism is alive, which has an independent genome, has a cellular membrane structure and a sustainable and autonomous replication system (Endy 2005; Toepfer 2005).

The autopoiesis theory acts on a philosophical assumption when developing a concept of life. Living organisms differ from non-living organisms by" [the fact that they are] the product of their organization […]. There is no separation between producer and product. Being and doing [of an] autopoietic unity is inseparable, and this constitutes their specific type of organization "(Maturana, Varela 1987). Consequently, the products of synthetic biology would not been self-caused, but organized externally by humans. Consequently, following this train of thought, scientists will never succeed in producing an autopoietic system synthetically. The human as manufacturer of the genome states himself as the creator of the organism. The postulated unit of producer and product can never be succeeded. The organism remains heteronomous by the determination of the genome by man.

This logical problem with the autopoiesis-theory in synthetic biology leads to restricted and modified applicability of the theory .

 

Thus, the following discussion will concentrate on the definition of life first illustrated and includes the autopoiesis theory in restricted form, as this will gain importance in synthetic biology for future developments.

After consideration of the machine concept and definitions of life it is evident that the organisms named "living machine" are by no means machines but living organisms. Today synthetically engineered organisms satisfy the minimum requirements of reproduction, metabolism and evolution. The term "machine" emphasizes the intended machine-like function. The organisms are alive but perform a machine-related purpose.

The claim of the synthetic biology is to create living organisms, which act simultaneously as a machine. The synthetic biology changes the definitions of living organisms and aims to abolish the separation between life and machine. It follows that the discussion with this term is not regarding the definition, but rather an ontological problem.

The term suggests a utilization of life with the pure focus on the control and goals of the humans.

 

On the one hand the term "living machine" generates an ethical problem, for the reason that it reduces living organisms to their purpose and implies a total control of created existence. Man breeds and keeps animals for the purpose of procuring food, which includes utilization and control of these creatures. There is an analogy to the organisms of the synthetic biology, however in synthetic biology humans appear as self-creators. Synthetic biology aims the creation of new organisms, in which the structures and properties are artificially defined by man whereas breeding and keeping animals is still subordinated through evolution.

 

On the other hand, "living machine" belies the fact that, due to a lack of understanding any intervention in the genome can indeed provide the desired functionality, but might also have not foreseeable effects. A feature of living cells is the interaction with the environment. It is difficult to assess the impact such interaction for the environment is as expected.

 

Furthermore, it has to be noted that currently it is possible to manipulate and utilize unicellular organisms, however in the future it is also conceivable to engineer higher organisms. The designation of "living machines" then abates these creatures to their pure function.

 

The concept of "living" and "non-living" organisms create a hybrid of both and does not answer which properties apply to these exactly. This might implicate associations about properties and facts which do not match the actual conditions in these organism. Furthermore the linguistic imprecision could have an effect on future ethical debates since it can open up possibilities for interpretation.

This has, under the assumption of a future "minimal cell" the result of changing perspective on life itself. The clear distinction between animate and inanimate is repealed, life as a laboratory adjustable process is demystified.

 

Artificial or artificially generated

-The „artificial cell“

An often used term in synthetic biology besides "living machines" is "artificial cell" or "synthetic cell". Both terms mean artificial, constructed, pseudo and pose as the opposite of "natural" (Oxford dictionary).

It should be noted what is exactly characterize as an "artificial" cell, as the term has no clear semantic boundaries. The laboratory-designed organisms are made as well as natural cells of organic material and have cellular structures. “Natural" cell carry the DNA or RNA necessary for reproduction, evolution, and metabolism information as well as the “artificial cell”.

The artificiality of the cells lies precisely in the laboratory completed synthetic production method, which is why they can be labeled as "genetic artificiality" (Birnbacher 2006).

 

The ambiguous and imprecise formulation can lead, as much as the phrase "living machine" to ethical problems. It is indicated that the synthetic modified organisms differentiate from "natural" organisms and are in contrast "unnatural". Splitting organisms in these two groups leads to an apparent dissection of the concept of life, even though both are "natural."

Again, as with the distinction between "living" and "non-living" this leads the division between "natural" and "artificial" to an altered prospect on life and a problematic classification and grading of the unit life (Boldt / Müller 2009 ).

 

Conclusion

Thinking ahead, the possibilities of creating new organisms through manipulation, reduction and modularization leads to man as a kind of "Creator". This opens up new ethical, social and philosophical areas of conflict. In this article the problem arising from the use of terms like "living machines" and "synthetic / artifical cell" were highlighted.

 

Particularly problematic is in our eyes, that there are several types of interpretation.

A science which has the possibility to improve the lives of people with new, low-cost and effective methods of environmental protection, the treatment of diseases and many other areas, should provide an unambiguous basis for an informed discussion. The fact that terms imply false and failing to reach full potential scenarios, prevents a qualified debate on opportunities and risks in this field. An example of this can be seen in the genetechnology debate, in which, through ignorance of the topic often irrational fears are raised which prevents a professional informed debate on this matter.

It is crucial to apply a definition, which is without ambiguity and reflects real possibilities and boundaries of this field. A proposal for a solution could be "mechanical living organism", through which the problem of the misleading hybrid character of terms is dissolving.

Referneces

 

Boldt J, Müller O, Maio G (2009) Synthetische Biologie- Eine ethisch-philosophische Analyse

 

Birnbacher D (2006) Natürlichkeit

 

Deplazes A (2011) The Conception of Live in Synthetic Biology

 

Deplazes A, Huppenbauer M (2009) Synthetic organisms and living machines

 

Endy D (2008) Reconstruction of the Genome, Science

 

European Commission (2003) Reference Document in Synthetic Biology

ftp://ftp.cordis.europa.eu/pub/nest/docs/synthtic_biology.pdf

 

Gibson D, Benders G, Andrews-Pfannkoch C,  Denisova E,  Baden-Tillson H,  Zaveri J,  Stockwell T,  Brownley A, Thomas D,  Algire M, Merryman C, Young L, Noskov V, Glass J, Hutchison III C, Smith H (2008) Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome

 

KurumaY, Stano P,  Ueda T, Luisi P (2006) A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells

 

Luisi P (2003) Autopoiesis: a review and a reappraisal

 

Matura H, Valera F (1984): Baum der Erkenntnis. Die biologischen Wurzeln des menschlichen

Erkennens

 

Pons Wörterbuch für Schule und Studium Latein; Hau; 2012

 

O’Malley MA, Powell A, Davies JF, and Calvert J (2008). Knowledge-making distinctions in synthetic biology. BioEssays

Oxford English dictionary http://www.oxforddictionaries.com

 

TESSY (Towards a European Strategy for Synthetic Biology)

http://www.tessy-europe.ec

 

Toepfer G (2005) Der Begriff des Lebens

 

Tucker R, Zilinskas J (2006) The Promise and Perils in Synthetic Biology

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)

 

[1] 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

[2] 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

[3] Stellungnahme der ZKBS zur Risikobewertung ecotroper C-Typ Retroviren der Maus, (Az.

6790-10-41, April 1996)

[4] 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

[5] 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

[6] 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

[7] Fields Virology 5th Edition (2007) Lippincott Williams & Wilkins

[8] RetroMax-System-Instruction Manual-IMGENEX

[9] 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

[10] The Journal of Gene Medicine Clinical Trial site

http://www.wiley.com/legacy/wileychi/genmed/clinical/

8

[11] Fehse B (2007) Insertionsmutagenese – Implikationen und Möglichkeiten der Vermeidung.

Schwerpunktprogramm 1230 der DFG

[12] 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)

[13] Methodensammlung der LAG (2009) Quantitativer Nachweis von Lentiviren (HIV1)-RNA

mittels Real time RT-PCR.

[14] Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (April 2009) Ringversuch

„Quantitativer Nachweis von Lentiviren (HIV1) RNA“-Ergebnisbericht

[15] Levy, JA (1995) The Retroviridae. Plenum Press, NY

[16] 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.

[17] 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).

 

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)	☺
General safety of the viral vector	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)	☺