Biosafety
Def: "biosafety measures aim to prevent the unintentional exposure to pathogens and toxins, or their accidental releas" 1
Risk assessment for our Adeno-associated virus based system
For the usage in the Federal Republic of Germany the Central Commission for Biological Security (ZKBS) released three legally binding Risk Assessment for the Adeno-associated Virus
26,
27,
28. The risk assessment in other countries could deviate from this decision, so please inform you about the legal regulation for the AAV in your country before using the Virus Construction Kit.
The classification in Biological Safety Level (BSL) is:
Adeno-associated Virus 2, 3 and 5 are to handle under BSL I
Adeno-associated Viurs 1, 4, 5, 7, 8, 9, 10 and 11 are to handle under BSL II
This classification was reasoned by the fact that only the serotypes 2, 3 and 5 are isolated from humans and it's harmlessness has been confirmed in clinical studies.
This general classification has to be rechecked when:
The Virus contains gene sequences with a transforming protential
For Viral Vectors that package a Vector plasmid that only contains the viral Inverted Terminal Repeats (ITRs) and provide the genes for the Rep and Cap genes in Trans as it is the case for our system:
These Viral Vectors are classified as BSLI if the following conditions are fullfilled:
The viral particles do not contain AAV derived sequences other than the ITRs
The viral particles do not contain Nucleotidesequences with a risk potential
We also investigated the legal regulations on AAV-2 Viral Vector systems in the United States. The guidelines of the National Institutes of Health (NIH) classifyes in the
Appendix B 32 AAV-2 Vector Systems as Risk Group 1 (RG1) Agents that can be treated under BSL 1.
adeno- associated virus (AAV) types 1 through 4; and recombinant AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus.
General specifications of the Adeno-associated Virus 2 (AAV-2):
In clinical studies the viral vectors were not dilivered to the gonades
Vectorsequences were not detectable in the patients blood or urine at an examination 48 h after an infection with AAV-229
In absece of Rep proteins the vector DNA stays extrachromosomal and is not frequently integrated.
Concluding all these informations and regulations the project that we have designed in this year is clearly to classify as BLS 1.
Legal regularisation in the Federal Republic of Germany
Which specific biosafety rules or guidelines do you have to consider in your country?
In Germany all working that includes recombinant DNA technologies is regulated by the Gesetz zur Regelung der Gentechnik. This law regulates general aspects arising from the life sciences and refers for more precise interpretations in §4 to the Zentrale Kommission für die Biologische Sicherheit. The ZKBS is a commission composed of 20 technical experts that releases yearly statements to actual issues of biosafety. So far the ZKBS released three stratements affecting the work with Adeno-associated viral systems
26,
27,
28. These documents were used to assess the dangers that could arise from our project to team members and the enviroment.
Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project?
At the Albert-Ludwigs-University Freiburg for all concerns of security the Stabsstelle Sicherheit is responsible and to contact if questions arise. Especially for questions of biological security Dr. Petra Markmeyer-Pieles is cognizant. We contacted her a first time befor the begin of our project in March when it was clear that the Adeno-associated Virus (AAV-2) was chosen as the topic of our project. At that time she proposed to do the cloning in the AAV-2 that is for sure to handle under biological security level 1 and to prepare everything for work under biological security level 2 to satisfy the precaution principle.
The precaution principle was realized and all viral vectors that contained a modified capsid were handled under SII conditions until proven harmless.
In August the planing of the project was completed, summarized in an Biosafety application30 and handed to the department for biological security who approve the application in an official BSL1 confirmation31official BSL1 confirmation for our project.
Risk management
Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?
Our project was designed in a way that it avoids any serious safety issues as far as possible.
When working with infectious particles a minimal risk for the researcher is allways present. This risk was minimized by restricting the transduced genes to fluorescent proteins and prodrug convertases that are already proven not to harm human cells in the absece of the corresponding prodrug. A potential danger for the public or the environment was minimized as much as possible by following strictly the rules of Good Laboratory Practice (GLP) and the abdication of using randomized insertions in the capsid and of replication potent viruses. Minimizing the risk for team members and the society was was allways one of the major concerns, especially because worries about undergraduate students manipulating a virus could arise.
The security concept will be explained by quoting and explaining the six guiding principles for safe manipulation of Gene Manipulated Organisms (GMOs) as summarized in Kimman et al. ; 200818.
1) Hazard recognition and identification
Risk assessment has been done and all legal regulations were considered as described in the last paragraph.
2) Biological containment
Biological containment means the usage of organisms with "reduced replicative capacity, inefectivity , transmissibility, and virulence"18.
For our project only replicative deficient viruses were used, additional all modifications aimed to have a specific targeting of the resulting viral vectors for a specific cell type. This gain in specificity requires it to cut off the braod natural tropism resulting is an less infective virus copared to the wild type virus.
3) Concentration and enclosure
All working steps for the preparation of viral vectors were carried out in a BSL II laboratory and within this laboratory work with the AAV was restricted to a separate Laminar flow cabinet type II. Cell culture and storage of the virus was also done in separate Freezers and incubators. All laboratories and epipment that contained viral vectors were specially marked, for example with a Biohazard warning signs.
4) Exposure minimization
This aspect of the guiding principles can be sumarized under "operator protection"18. The Exposure minimization was achieved in our laboratory by wearing special labcoats for the SII laboratory and gloves that were desinfected and changed regualrely. During manipulation of viral vectors attention was payed to avoid droplets and especially aerosoles. A possible diversion of the viral vectors was avoided by cleaning all equipment when inserted or removed from the Laminar flow cabinet and after completion of the work task.
5) Physical containment
The requirements for the physical containment were fullfilled by performing all manipulation on the AAV in an BSL II laboratory that guaranted a restriction of persons that entered the laboratory.
6) Hazard minimization
For the AAV-2 there are no sugestive activitis because the possible danger that runs out of the AAV is comparably low, vaccination is not avilible and biomonitoring is not necessary.
Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?
Several composite parts that were assembled by our Team this year are alone capable of producing infectious viral particles when transduced together with a vector plasmid and a helper plasmid into AAV-293 cells. These special cells provide the adenoviral gene E1 stabily integrated in trans.
These cells are not provided in the Virus Construction Kit nor availible in the Parts Registry and have to purchased from other laboratories or a commercial supplyer.
For this reason we estimate the risk of a accidental transformation of AAV-293 cells with all three plasmids for negligible.
Nevertheless we considered it useful to mark every BioBrick or Composite Part in the Registry that contributes to the production or is capable of producing viral vectors when transformed under the previously mentioned conditions.
Contribute to community discussions on what needs to go into a code against the use of our science for hostile purposes (see A Community Response)
Public Perception
importance of maintaining public legitimacy and support
apllications should demonstrate clear social benefits
not overhyped - anxiety and unrealistic hopes
psychological research into the concept of "identity-driven decision-making" (Torpman,2004) 19
Every grout has a set of norms: a code of conduct about what is acceptable beahviour (Jaques, 2004] 19
Trade-off between potential misuse and promising medical progress
Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?
In principle each research-project that bears any risks for engaged researchers, mankind or the environment should be treated under the precautionary principle as proposed 11: "treat synthetic microorganisms as dangerous until proven harmless".
This would mean to work on such synthetic DNA containing Bio Bricks at least under Biological security levels two.
Additional to this secure working environment the system itself can be optimized according to biosafety aspects, means to reduce it's viability outside the laboratory. This aim can be approached by reducing the systems ability to evolve, proliferate and interact with it's environment. A common method to achieve this goal is to engineer microorganisms in a way that they depend on nutrients that can't be found in the environment in sufficient amount.
Biosecurity
Def: "measures focus on the prevention of theft, misuse , or intentional relese of pathogens and toxins"
1
The malignant use of biological agents in history
The misuse of biological agent as weapons in warfare is a fear spreading companion in the history of mankind, ranging from the well-poisoners in prehistoric times to bio-terrorists present days. The following brakt intends to give a short outline of the major events[http://www.ncbi.nlm.nih.gov/pubmed/12789408 22]:
First systhematic use of chemical weapons during the first world war
The 1918 flu pandemic lasted from March 1918 to June 1920 and killed approximately 50 Million people around the world. Rumors circulated that this pandemic is caused by the other combatant nation.
"Prohibion of the Use of Asphyxiating, Poisonous or other Gases and of Bacteriological Methods of Warfare" was signed on 19 June 1925[http://www.ncbi.nlm.nih.gov/pubmed/12789408 22]
Several combatant nations of the second world war established biological warfare programs.
The range of the Japanese biological warfare program lead several nations to expand their own biological warfare program
Limited military use22 and the advances in molecular biology (e.g. the first isolatin of a gene and the discovery of the restriction enzymes in 1969) lead to the "Convention on the Prohibion of the Development, Production and Stockpilling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction " (BTWC) on 10 April 1972.
''"develop, produce, stockpile or otherwise acquire or retain: ... Microbial or other biological agents, or toxins whatever their origin or method of production, of types and in wantities that have no justification for prophylactic, protective or other peaceful purpose..." ''3
Breach of the BTWC by the Soviet Union which continued their offensive biologicla warfare programm
Stop of the biological warfare Program "Biopreparat" accompanies the Dissolution of the Svoviet Union in 1991
Antrax attacks in the USA in 2001
After this short description of misused biological components there is still the question what impact biological warfare will have in the future of mankind.
Broad avilibility of knowledge
Broad avilibility of material
Szenarios of attack
Conclusion
Include something in your project description and presentations that demonstrates that you have thought about how others could misuse your work.
For sure there is allway the possibility that knowledge to produce transgene viral vectors could be used to produce bioweapons. Therefor it was important for us to use a system that does not bear the risk that someone could use it for evil purpose. In the case of the Adeno-associated virus the very limited packaging capacity is the major reason that excludes it from the list of agents that could realistically be used for the pruduction of bioweapons. Even a fully replication potent AAV will depend on the coninfection of a helpervirus and is therefore not suitable for a fast propagation in an population.
Additional to this point we concentrated our project on the retargeting of the virus - means to make the broad tropismn more narrow and to decrease the transduction efficiency in the most cases. This modification is usually mainly required for medical purposes. Also we did neither investigate possibilities to shield the vector from the immune system of potential host nor ways to bypass an existing immunity.
01 Synthetic Biology & Biosecurity - Awareness in Europe | Kelle ; 20071
02 Synthetic Biology - Social and Ethical Challenges | Balmer et Martin ; 20082
03) Synthetic Biology - Applying Engineering to Biology
used
04 The Impact of the Development of Modern Biology and Medicine on the Evolution of Offensive Biological Warfare Programs in the Twentieth Century | Dando ; 1999 04
05 Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template | Cello et al. ; 2002 05
06 Dangerous research : When science breeds nightmares | Steinbruner et Harris ; 2003 06
07 Synthetic Genomics - Options for governance | Garfinkel et al. ; 2007 07
08 Synthetic biology | Benner et Sismour , 2005 08
09 Synthetic biology—putting engineering into biology | Heinemann et Panke ; 2006 09
10 Revealed: the lax laws that could allow assembly of deadly virus DNA | Randerson ; 2006 10
11 The Promise and Perils of Synthetic Biology | Tucker & Zilinskas ; 2006 11
12 1918 Flu and Responsible Science | Sharp ; 2005 12
13 The Darker Bioweapons Future | CIA ; 2003 13
14) [[Media:Primer for Synthetic Biology - Mohr 2007.pdf]]
15) [[Media:Freiburg10_The bugs of war.pdf]]
16 Expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox. | Jackson et al. ; 2001 16
17 Governance of dual-use research: an ethical dilemma. | Selgelid ; 2009 17
18 Evidence-based biosafety: a review of the principles and effectiveness of microbiological containment measures. | Kimman et al. 2008 18
19) A Hippocratic Oath for life scientists | Revill et Dando ; 2006 19
20 Empowerment and restraint in scientific communication. New developments make it easier to share information, but more difficult to deal with dual-use biology. | Campbell ; 200620
21 When risk outweighs benefit | Aken ; 2006 21
22 Advances in life sciences and bioterrorism. Risks, perspectives and responsibilities.| Beck ; 2003 22
23 PNAS policy on publication of sensitive material in the life sciences | Cozzarelli ; 2003 23
24 Characterization of the
Reconstructed 1918 Spanish
Influenza Pandemic Virus | Tumpey et al. ; 200524
25 Deadly flu virus can be
sent through the mail| v. Bubnoff; 200525
26 Risk assessment of human Adeno-associated viruses| ZKBS; 200126
27 Advises for AAV carrying cell cycle regulating genes| ZKBS; 2004 27
28 Risk assessment of human Adeno-associated viruses and AAV derived vectors| ZKBS; 2005 28
29 Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.| Kai et al. ; 2000 29
Bioverteilung in Klinischer Studie
30 Biosafety application of the iGEM team Freiburg_Bioware 201030 (in German)
31 Official classification as Biological Safety Level 1 by the local biosafety office31
32 Appendix B | National Institute of Health 32