Introduction

The full definition of bioweapon (later referred to as BW) as given by the Merriam-Webster online dictionary is as follows:

"a harmful biological agent (as a pathogenic microorganism or a neurotoxin) used as a weapon to cause death or disease usually on a large scale" (1)

This is a broad definition including naturally occurring toxins and pathogens, as well as those specifically engineered to cause disruption and/or death. However, biological warfare is usually discussed as distinct from nuclear and chemical warfare, which together form the triumvirate of terror given by the military acronym NBC. A bioweapon may be lethal or non-lethal, in the latter case having, for example, a primary purpose of debilitation, and may be targeted at individuals, small groups or entire populations. Associated phrases include biodefense, biowar and bioterrorism. (2)

The distinction between chemical and biological agents is sometimes subtle; for example, toxic products of living organisms, such as ricin, may be considered dangerous chemical agents, where the organisms themselves, in this case the castor oil plant, do not qualify as the biological equivalent. This is evident from the fact that we produce more than 1 million tonnes of castor beans every year. All such weapons have the potential to be labelled as weapons of mass destruction, or ‘WMD’s. (3)

A Short Historical Account

The use of biological agents as a tool of war has been with us since antiquity. Its most significant initial use in modern times was to enforce colonialism; for example, smallpox was likely disseminated by the British Marines to quell opposition to their settling of New South Wales by the native population in 1789. Similarly, smallpox-infected blankets were handed out to Native Americans by the British in the 1760s, including in times of peace. (4)

The devastating power of bioweapons was recognised properly at the beginning of the 20th century, with the advent of international agreements that criminalised certain wartime behaviours, called the Hague Conventions (in 1899 and 1907). These regulated the use of BW via a clause prohibiting signatories from using projectiles with the capacity to spread ‘asphyxiating poisonous gases’. All major powers ratified this doctrine except for the United States, infamous for unilaterally eschewing international regulation. After the Great War, it was felt that further clarification of this point was needed, perhaps given the extensive use of mustard gas by the Germans, and this resulted in the Geneva Protocol of 1925, which extended the Hague Conventions to prohibit generally the use of ‘bacteriological methods’ in warfare (signed by the US 50 years later). (5,6)

There are many criticisms to be levied at this agreement, most specifically its lack of application to non-ratifying countries, and failure to prohibit the stockpiling of BWs and their use in civil/domestic contexts. This protocol did not, for example, prohibit the extensive development and weaponisation of toxins by the Allied forces in World War II, including but not limited to tularemia, anthrax, brucellosis, and botulism. We are fortunate that these were never deployed, largely due to the threat of retaliation which made it strategic suicide, similar to the ‘mutually assured destruction’ reasoning prevalent during the Cold War. Nevertheless, the Geneva Protocol is now considered part of so-called ‘customary international law’; that is, law that should be observed by all responsible and right-acting international actors.

Furthermore, biological and chemical warfare has been a constant spectre in the Middle East in recent decades, from Saddam Hussein’s chemical assault on Kurdish rebels at Halabjah in ’88 to the euphemistic Gulf War Syndrome with which US and Coalition survivors of the first Gulf War were diagnosed in ‘92 (likely a result of the contaminants released from various Iraqi chemical plants and stockpiles when subject to airstrike). As recently as 2013, the Assad regime in Syria was accused of killing hundreds of Syrians in Ghouta using sarin gas. My country, the UK, was guilty of issuing export licenses for chemicals for sarin in January of that year.

It is not just the context of warfare in which this discussion must be had. Another case of the deliberate misuse of biological agents was the 2001 anthrax attacks in the US, in which envelopes containing anthrax were delivered to various news offices and Senators, killing five people. In other scenarios, agents are not introduced maliciously but can develop into an epidemic, as with the ebola currently gripping West African nations like Sierra Leone and Liberia, which is exacerbated and perpetuated by events like the theft of infected materials. Malevolent forces may try and harness or reproduce outbreaks of diseases like ebola for ‘evil’. For example, evidence surfaced recently that ISIS, the Islamist militia group, had been trying to weaponise the bubonic plague. (7,8,9,10)

All of this compounds to demonstrate the devastation which anyone capable of engineering and disseminating particular biological agents could inflict upon a population. Synthetic biology, whilst pushing the scientific envelope and extending the frontiers of our ability to manipulate the world around us, creates a unique problem by opening up another avenue for the abuse and ill-purposing of science. With minimal expenditure and an education available entirely online, your average Joe can theoretically build a biological weapon of mass destruction.

Safeguarding our Project Against Misuse

Given this summary and line of reasoning, we decided it would be pertinent to try and assess the potential for our replicon and for our general theory to be co-opted and abused. The intention of our work is to further the cause of modern medicine, reinvigorate the fight against diabetes, and inspire a new line of research into all things RNA. We do not want it to be used for the advancement of a military agenda or for murder, so it is in our interests to evaluate the possibility of it being hijacked and reengineered against its stated purpose. To this end, we had a conversation with Dr Robert Spooner of the University of Warwick, who has experience working with ricin, to try and establish some possible safeguards, which I now go on to describe.

The first and most obvious way our design might be twisted is with the replacement of the target siRNA code, which in our version is determined from DPP-IV since this is what we’d like to downregulate the expression of, with code from a gene that produces some vital enzyme or other essential protein. This is already recognised as an incidental danger in the genetic modification of plants for which the method of modification is based on RNA interference, just like our project. In one example a scientist in New Zealand identified that the genes disrupted in a modified strain of wheat, SEI and SEII, are shared to a high degree by humans, and also asserted the ability of the culprit dsRNA strands (which would later form siRNA strands) to survive cooking and processing of the wheat and the gastrointestinal processes associated with human digestion, to make it into the blood stream! This is a case not of bowdlerizing the replicon, but of hijacking its most essential function. In its most extreme and terrifying form, this idea extends to eugenics because some populations may have genes vital to their survival that others do not. (11)

The other problem concerns harnessing the power of self-replication inherent to the replicon to persistently produce a harmful toxin within a person’s cells. As opposed to the previous problem, this represents a complete subversion of the replicon’s intended function, repurposing it as a constant producer of toxin rather than a platform from which to launch RNA interference.

Our response to these problems is multifarious. Firstly, we have implemented an aptazyme kill switch in our replicon, so that specific exogenous compounds are able to seek it out and destroy it. This means that if it was adapted as a chassis for some dangerous choice of siRNA, by cutting out and replacing that 25bp part of the genetic code, but it still contained the aptazyme, we could deliberately target and destroy it in vivo to stem the tide. We have demonstrated this via experiment, and it is an essential safety mechanism. Further to that, cutting out the siRNA element and then ligating together the resulting parts is an unwieldy and difficult process which requires intimate knowledge of the construction of our replicon; needless to say, there are perhaps more expedient ways of creating a biological weapon.

Similarly, an attempt at swapping out the aptazyme for some other RNA cargo would likely fail. Efficiency issues would arise because of the regulatory MS2 box, which would dampen exponential replication.

In response to the toxin question, for this to be an effective attack the toxin would have to build up in a cell in a significant quantity if it were to do any damage. The rate of translation of the RNA strand would therefore have to be high enough to meet this demand. This would require both a more efficient RdRP, which will likely have to be arrived at by random mutagenesis, which would be a severely arduous task, as well as the complimentary promoters working in parallel.

In conclusion, we do not deem the threat to be significant, but nevertheless significant and meticulous structures need to be in place to properly regulate synthetic biology, for researchers, hobbyists and industry alike.

Crude Bibliography

1. Merriam-Webster dictionary definition
2. Wikipedia entry on bioweapons
3. Wikipedia entry on ricin
4. Counterpunch article on the history of bioweapons
5. Wikipedia on Hague Conventions
6. Wikipedia entry on Geneva Protocol
7. Wikipedia entry on 2001 US anthrax attacks
8. Ebola distribution map from the CDC
9. Huffington Post article on theft from ebola clinic
10. Vox article on attempted weaponisation of bubonic plague by ISIS
11. Paper on RNA interference in GM wheat by Jack Heinemann