Biosafety

Definition: "biosafety measures aim to prevent the unintentional exposure to pathogens and toxins, or their accidental release" ¹

Risk assessment for our Adeno-associated virus based system

In Germany the "Central Commission for Biological Security" (ZKBS) released three legally binding Risk Assessment satements for the Adeno-associated Virus ^{26, 27, 28}. Risk assessment in other countries may deviate from these decisions, so please inform yourself about the legal regulations on AAV in your country before using the Virus Construction Kit.

• Adeno-associated Virus 2, 3 and 5 have to be handled under BSL 1.
• Adeno-associated Viurs 1, 4, 5, 7, 8, 9, 10 and 11 have to be handled under BSL 2.

This classification was developed based on the fact that only serotypes 2, 3 and 5 are isolated from humans and that their harmlessness has been confirmed in clinical studies. This general classification has to be rechecked when the virus contains gene sequences with a transforming protential.

Viral vectors systems packaging a vector plasmid that only contains the viral Inverted Terminal Repeats (ITRs) and providing the genes for Rep and Cap in trans (as it is the case for our system) are classified as BSL 1 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 for AAV-2 Viral Vector systems in the United States. The guidelines of the National Institutes of Health (NIH) classify in the Appendix B³² AAV-2 Vector Systems as Risk Group 1 (RG1) agents that can be treated under BSL 1. In detail the appendix states "... 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."

• In clinical studies the viral vectors were not dilivered to the gonades.
• Vector sequences were not detectable in the patients blood or urine at an examination 48 h after an infection with AAV-2 ²⁹.
• In absence 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 classified as BLS 1.

General biosafety regulation in Germany

Which specific biosafety rules or guidelines do you have to consider in your country?

In Germany all work that includes recombinant DNA technologies is regulated by the Gesetz zur Regelung der Gentechnik. This law regulates general aspects in 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 events²²:

• 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 1925²²
• 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

n the life sciences information has allways been freely acessible for everybody who is interested in the results of a particular research project. The combination of this global availibility with the new possibilities of the internet broad for literature search and availibility of gene sequences made it easiere to collect informations for a possible misuse.
For the overwhelming majority this open availibility is absolutely desirely but on the other hand there are also examples of research results that bear a very high risk to be misused.
Following we present the three most controversial discussed publications that could also be read as a "How to create your own bioweapon".

Mousepox Virus in Australien

The Australian research group around Jackson et al. ; 2001 ¹⁶inserted the coding region of the IL-4 gne into the genome of mousepox. They hoped to create a virus that sterilizes mice and thus provides a means for pest control. Contrarely to their expectations they had created a superstrain that killed even naturally resistent mice and mice that had been vaccinated against normal mousepox. This discovery could potentially be used to make smallpox resistant to potential vaccines.

Synthesis of a Polio Virus

At the State University of New York Cello et al. ; 2002 ⁰⁵ synthesized a "living" polio virus from scratch. Especially the Supporting Online Material caused worried remarks because it precisely describes how to assemble a virus from small oligo nucleotides. The authors jusifyed their project by declaring that they:

"made the virus to send a warning that terrorists might be able to make biological weapons without obtaining a natrual virus"¹⁷

Reconstitution of the Spanish Flu

At the Center for Disease Control and Prevention (CDC) the group around Tumpey et al. ; 2005²⁴ published that they had sequenced and recreated the pandemic Spanish Flu Virus of 1918 which killed 20-50 million people. The publication of the sequence provoked several very different responses that can only be partially be quoted here. The controversial noticed outrider of the Synthetic Biology Craig Venter seen in the new virus

"the first true Juressic Parc scenario" ²

The Institute Professor at the MIT Philip A. Sharp supported the publication because he:

"belive[s] that allowing the publication of this information was the correct decision in terms of both national security and public health" ¹²

Wheras v. Bubnoff; 2005²⁵ critisizes the CDS for it's careless regulations for the shipment of viruses and the willingness to propagate highly virulent viruses. This causes in his opinion the risk of possible accidents resulting in the release of the virus to the environment.

Availability of molecular biological techniques

The knowledge required for the creation of a genetically engineered virus can easily be accessed from all over the world in online gene banks and publication databases and could be potentially be used for a destructive purpose ²⁰. In 2007, Scott C. Mohr published the first part of his manuscript Primer for Synthetic Biology¹⁴. This open accessible document explaining the basics of molecular engineering addresses people interested in genetics but coming from a non-academic background. This development called "garage biology" or "biohacking" is a clear indication for the tendency to have knowledge about molecular biology available in society, as it was seen with programming and computer hacking from ~1980 on.

Availability of synthesized DNA

One of the reactions to the publication of the genome sequence of the Spanish flu strain from 1918 was v. Bubnoff, 2005²⁵ who pointed out the lax handling of the reconstituted virus sequence, the ease of availability and the low effort that has to be invested to recreate a virus:

''"Scientists in Canada are planning to work with the virus, although they will not request it from the CDC. [...] Reconstructing the live virus from its DNA would then take just a few days, he says.''

Technical advances and dropping prices in the field of gene synthesis brings several deadly germs into the range of small research projects or even private persons as for example Spanish Flu (Influenza H₁N₁) with a 13.5 kbp ^* or even the Ebola virus with a 19 kbp genome ^*. In 2006, the “Guardian” journalist James Randson ordered a 78 bp DNA oligonucleotide coding for the capsid of the smallpox Orthopoxvirus variola with the intention to alert the public. ¹⁰
In the United States, smallpox are listed as schedule 5 pathogens in the Anti-terrorism, Crime and Security Act of 2001 and are illegal to keep or use without first notifying the civil authorities.
On the other hand, it is questionable if these prohibitions or perhaps the size of the smallpox genome of ~200 kb ^* are sufficient to prevent de novo synthesis in the future efficiently. His intention was to focus the public's attention to the possibility that one of the most severe plagues in the history of mankind could be synthesized and released to the environment, either intentionally or by mistake. This would be a tragic setback since the smallpox's eradication was celebrated in 1979 by the World Health Organization as one of the greatest victories in the history of medicine ³³.
In order to prevent the unauthorized synthesis of gene sequences encoding hazardous biological agents, efforts to implement sequence analysis algorithms into the operating procedure of all organizations and companies capable of synthesizing gene sequences are being undertaken.

This barrier should be implemented not on a voluntary basis but as a legally binding regulation. It should be enforced by the government on a national level, but effort should also be made to find solutions for an international progress on this issue.
As a first step, the development of search algorithms as Craic's BlackWatch²² Several countries around the globe are technically capable to develop biological weapons or to support terroristic groups in doing so. When this physical capability meets an ideological attitude that is based on contempt for other concepts of society, it cannot be ruled out that biological warfare is taken into consideration.

Synthetic Biology vs. Nuclear Research - Consideration in terms of security policy

When considering the history of research, the dual-use character of technical innovations has always been present, only the reaction of society to it differs from case to case. As an example, scientific discoveries in the field of nuclear physics where treated as classified information having a high relevance for the national security as shown in the case of the Manhattan project¹⁷. A comparable censorship is not established in life sciences research apart of some cases when national security was endangered.

Another point concerning the threat arising from biological weapons is the absence of methods that can be used to monitor such weapons of mass destruction as pointed out by M.R. Dando. This fact and the increasing world-wide mobility would make it impossible to prevent the spreading of such weapons when once accessible to potential assassins.

Is a revision of basic research necessary in sensitive research fields?

The publication of research papers containing dual-use knowledge caused several people to call for regulation. In the United States, the so called Fink Committee evaluated the possibility that research in life sciences could be used for biological warfare purposes and how this could be avoided.¹

This committee had two recommendations:

• To familiarize the scientific community with the dual-use problem in the life sciences.
• To review experiments of concern. Experiments are of concern if they:
  • render a vaccine ineffective
  • confer resistance to therapeutically useful antibiotics or antiviral agents
  • enhance the virulence of a pathogen or render a nonpathogen virulent
  • increase transmissibility of a pathogen
  • alter the host range of a pathogen
  • enable the evasion of diagnostic/detection tools
  • enable the weaponization of a biological agent or toxin
• To review publications with a strong dual-use factor
• To create a National Science Advisory Board
• To improve the oversight and reduce unauthorized accessibility of hazardous gene material
• To include the life sciences into the efforts for national security
• To harmonize the international oversight over dual-use research

The implementation of these recommendations would be desirable, even though they might cause inconveniences for scientists working in the affected fields.

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.

Concluding all factors mentioned above, we need to consider the possibility that a person, organization or state could misuse the fast-advancing life sciences for biological warfare. The possibilities and the simplicity of dual-use research misuse will become more and easier the faster the scientific progress advances.
Therefore it is important to minimize these potential risks before they become reality. This is not only the task of a designated group but a moral obligation for scientists, politicians and everybody related to dual-use research. Especially scientists have to contribute to and lead the continuing discussion on this topic because they are able to estimate how aspects of their research fields might be misused.

• 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 Primer for Synthetic Biology | Mohr ; 2007 ¹⁴
• 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
• 33 Media centre - Smallpox | World Health Organisation ³³
• 34 BlackWatch Homepage | Craic computing³⁴