Weapons of Mass Destruction, do the WHO Proliferate?

 Sarah Westall Show Today: Millions of Gates Mosquitoes on 12 countries. Yours is next

Draft prepared for the Institute for responsible technology 2021.

Bio Technology as Bio Weapons

Rapid Advances in Biotechnology: Implications for Bioweapons Development

 

 

 

Contents

 

Introduction

Biotechnology of Bioweapons

Dual Use Research

Gain of Function Research

Biological Toxins and Gene Editing

In Vivo Genetic Modification

Synthetic Biology: Bioweapons Application

Multiplex Automated Genome Engineering (MAGE)

Conclusion

References

 

 

Introduction

 

Rapid developments in genetic engineering have recently created unique challenges: Infant technologies are being used in multiple settings to modify and generate microorganisms, aiming to promote particular traits. The accidental release of a novel species or pathogen could have far reaching environmental, health, social, and economic consequences but there is also the risk of the intentional misuse of biological agents. National and international security risks have significantly expanded due to the accessibility of technology with the capacity to create new biological weapons. These weapons are designed for a new type of warfare and conflict, as they can be deployed covertly. Nonetheless, bioweapons are potentially more deadly than nuclear weapons and are internationally recognized as a weapon of mass destruction. 

 

The military point to their usefulness first:

“The purely financial advantage of employing biological weapons was clearly illustrated by a 1969 expert United Nations panel which estimated the cost of operations against civilian populations at $1/Km² for biological weapons, versus $600/Km² for chemical, $800/Km(2)² for nuclear, and $2,000/Km² for conventional armaments” FAS, 2021

There are many different ways genetic engineering can create potentially dangerous bioweapons:

-       impart antibiotic resistance to bacterial pathogens

-       increase human-to-human transmission of a deadly pathogen

-   make an existing pathogen resistant to existing vaccines, therapeutics, or other countermeasures.

However, there are also some important conditions an effective biological weapon has to meet:

-    Procurement: one has to be able to procure a suitable biological agent. “The NATO definition of a biological agent is: A microorganism (or a toxin derived from it) which causes disease in man, plants or animals or which causes the deterioration of material.” They include Bacteria, Viruses, Rickettsiae, Chlamydia, Parasites, Fungi and Toxins.

-      Mass production: a bioweapon needs to be produced at scale without the loss of pathogenicity,  Making microorganisms effective as self-propagating.

-       Effective delivery: a biological agent typically needs to be deployed quickly and en masse to achieve its maximum impact before effective countermeasures can be deployed.

-   Environmentally robust: a biological agent needs to be able to survive in the environment and spread.

-    Treatable: the agent that releases a biological agent needs to have an effective countermeasure to prevent incurring damage from the biological agent. 

In “Guidance Notes to the Field Commander on the Consequences of Individual Agents For Continued Operational Effectiveness”, they include over 40 examples of biological agents, for example, Epidemic Typhus and Pneumonic Plague. (https://fas.org/nuke/guide/usa/doctrine/dod/fm8-9/2appc.htm).

 

Most naturally occurring pathogens do not fulfill all of these conditions, making them unsuitable for use as a bioweapon, where genetically engineered pathogens with enhanced functions could.  

Recent times have seen the rise of non-national agents that might not consider the last condition (that treatment is available) to be necessary, as they would tolerate inflicting damage to their population if that meant inflicting much greater damage to their intended targets. This of course goes for our hostile use of biological weapons on other countries as well. 

Dissemination includes inhalation, dermal exposure and ingestion. Therefore contamination of the food supply has been described as the weaponization of agriculture. (https://apnews.com/article/genetics-science-biological-weapons-ap-top-news-us-news-8ed74d87df524ab580d7fbd3b845d0c6)

Meeting these conditions has been the main historical limiting factor in the development and proliferation of biological weapons: Very few countries were able to develop biological agents and the means to effectively deploy them. In addition, some deadly pathogens, like smallpox, exist only in high-security laboratories, and most potentially malicious actors would find it difficult or impossible to procure them. 

New gene editing techniques developed in the biotechnology industry provide an opportunity for malicious actors to procure and manufacture bioweapons: It is now possible to synthesize a pathogen from a digital genetic code from an online database. Genetic engineering processes no longer require highly trained and experienced staff and specialized equipment. This has significantly expanded the scope of malicious actors who could feasibly obtain and manufacture diseases which were contained or eradicated decades ago. In response, there have been consistent calls from the scientific community and the broader public to develop more effective methods of detection and prevention (Fraser, 2001).

 

Biotechnology of Bioweapons

 

The advances in the field of biotechnology have made genetic engineering much more accessible than ever before. The past several decades have seen the development of increasingly sophisticated means of editing a genome: First, there were molecular techniques like meganuclease, transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs), then small RNAs techniques, including microRNA (miRNA) and small interfering RNA (siRNA) followed, and finally, CRISPR/Cas9 has recently become the most dominant and most reliable genome editing technique (Khan, 2019).

“At the moment, most gene editing involves "Crispr" – a set of genetic scissors first developed by the Nobel-prize winning scientists Emmanuelle Charpentier and Jennifer A Doudna in 2012. The technology relies on a kind of ancient immune system found in a large number of bacteria. When they encounter a potential viral threat, they copy and paste some of its DNA into their own genome, then use it to develop a pair of scissors that can identify that exact sequence. If they ever meet it again, they simply snip, and deactivate it.” (Gorvett, Z. 2021) 

Later Gorvett disputes CRISPR’s reliability and ability to deactivate an exact sequence, referencing studies claiming more than 50% of genetic engineering experiments observed unexpected and unwanted mutations.

Figure 1. Overview of genome editing techniques (Khan, 2019)

Over the past few decades, gene editing technologies have not only grown significantly in variety and availability (Figure 1), they have become much more accessible. CRISPR technology, in particular, has reduced the cost of the gene editing process. For $990-1990, one can acquire a complete CRISPR gene editing kit online capable of performing gene knockouts, point mutations, and gene knock-ins. This has made gene editing technology widely available to people with little or no knowledge of genetics (Scientific American, 2017). In 2017, German authorities in Bavaria found that two DIY CRISPR bacteria kits contained potentially pathogenic bacteria. Although the risks of creating a dangerous pathogen using these DIY kits are considered to be relatively small, the wide availability of these kits, complete lack of regulation and oversight, and their potential widespread use certainly raise national security questions. Although these kits alone would not be sufficient to create a homemade bioweapon, they most likely make this enterprise easier for a potential malicious agent with sufficient knowledge. Most importantly, they are currently not regulated nor their sale monitored by the relevant agencies.

 

Dual Use Research

 

Dual-use items are goods, software and technology that can be used for both civilian and military applications” (EU Dual Use Trade Controls: https://ec.europa.eu/trade/import-and-export-rules/export-from-eu/dual-use-controls/)

The 1925 Geneva Protocol (or the Protocol for the Prohibition of the Use in War of Asphyxiating, Poisonous or other Gases, and Bacteriological Methods of Warfare) and the 1972 Biological and Toxin Weapons Convention legally limited the development, production, acquisition, transfer, stockpiling and use of biological weapons. However, the absence of meaningful mechanisms for ensuring that countries comply with their obligations remains a major weakness. (https://www.amacad.org/publication/governance-dual-use-technologies-theory-and-practice/section/5) . The Geneva Protocol banned the use, but not development or production of biological weapons, while the 1972 Biological Weapons Convention specifically prohibited their development, production, and proliferation. This established a strong global norm on biological weapons, which effectively established a worldwide ban. This was further augmented by the 1993 Chemical Weapons Convention (or the Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on their Destruction), which planned but failed to introduce extensive verification measures, like on-site inspections. 

Therefore, when the Chemical Weapons Convention became effective in 1997, a global ban on biological weapons came into effect. This meant that no country, including the US, was allowed to develop biological weapons. However, this applies to any research with a specific goal to develop a bioweapon. It does not apply to research where bioweapon development can occur as a side-product. There is research that produces results that can be applied to weaponizing biological agents, and where the research produces organisms that can be utilized as bioweapons. The results of this research can, therefore, have multiple uses, one of which is a bioweapon.This is how dual use research is so far not illegal. 

However, two phrases are inextricably linked to bioweapons research: “dual use research of concern” (DURC) and “gain of function.” 

The National Institutes of Health define DURC as: 

“Dual Use Research of Concern (DURC) is life sciences research that, based on current understanding, can be reasonably anticipated to provide knowledge, information, products, or technologies that could be directly misapplied to pose a significant threat with broad potential consequences to public health and safety, agricultural crops and other plants, animals, the environment, material, or national security.” (NIH, 2021a)

Therefore, DURC includes not just human pathogens, but also some plant and animal pathogens, because these could, if released accidentally or intentionally, cause significant economic and environmental damage. The following criteria are used to evaluate whether research can be classified as DURC:

“Can the research be reasonably anticipated to produce one or more of the seven experimental effects/categories listed below?

Will an intermediate or final product of your research make a vaccine less effective or ineffective? YES / NO

Will the final or intermediate product of your research confer resistance to antibiotics or antivirals in ways that are inherently different from those published previously? YES / NO

Will your work enhance the virulence of a pathogen or render a non-pathogen virulent? YES / NO

Will the results of your work increase the transmissibility of any pathogen? YES / NO

Will your research result in alteration of the host range of a pathogen? YES / NO

Will your research result in a product or intermediary that may prevent or interfere with diagnosis of infection or disease? YES / NO

Does your research enable “weaponization” of an agent or toxin? YES / NO

Even though your research did not involve any of the aforementioned seven criteria, and recognizing that your work product or results of your research could conceivably be misused, is there the potential for your results/product to be readily utilized to cause public harm? YES / NO” (NIH, 2021b) 

If the answer to any of these questions is YES or potentially YES, then the research could potentially be DURC and is reviewed and assessed by the Institutional Biosafety Committee (IBC) to determine whether the criteria for DURC is met. If research is classified as DURC, any publication of its results has to be authorized by the NIH Deputy Director for Intramural Research's Dual Use Committee, to make sure that sensitive or dangerous information is not released into the public sphere.

However, communication of research results and sharing the benefits to society are required both to a) justify conducting dual use research at all and b) provide information to do a meaningful risk assessment. Prohibiting the publication of results also protects organizations and laboratories from scrutiny by regulators, in regard to biosafety and national security during export, transfer, brokering and transit of dual use items. (https://trade.ec.europa.eu/doclib/docs/2020/january/tradoc_158576.pdf)

 

“The Biological Weapons Convention (BWC) was the first international treaty outlawing an entire class of weapons of mass destruction. From the outset, the BWC’s terms acknowledged the dual-use nature of biological agents: instead of prohibiting biological weapons specifically, it committed parties never to “...acquire or retain: microbial or other biological agents, or toxins . . . of types and in quantities that have no justification for prophylactic, protective or other peaceful purposes,” as well as “weapons, equipment or means of delivery designed to use such agents or toxins for hostile purposes.”⁵ This language also ensured that the BWC’s fundamental prohibitions would apply to all future scientific and technological developments in the life sciences and related fields, including in the nascent field of biotechnology.

 

At the same time, the convention commits its parties to facilitate the fullest possible exchange of materials, equipment, and information for using biological agents and toxins for peaceful purposes and to avoid hampering international cooperation in such activities. This tension between the nonproliferation and assistance provisions of the BWC has been a major source of controversy between developed and developing countries since the earliest days of the convention. https://www.un.org/disarmament/biological-weapons/

 

Gain of Function Research

 

Gain of function (GOF) research is a type of research where natural mutation processes of viruses and bacteria are accelerated in a laboratory to impart enhanced traits to these organisms. The objective is to create pathogens with increased human-to-human transmission, virulence, antigenicity, or antibiotic resistance. This research is done to identify specific mutations that lead to these traits, so they can be monitored in the wild pathogen population, or used to develop countermeasures. It is easy to see how developing pathogens that have more dangerous traits than their naturally occurring counterparts is highly dangerous and can be considered bioweapon research.

In addition to accelerating natural mutations in a laboratory, there have been several relatively recent cases of engineered pathogens that would be much more dangerous than their naturally occurring counterparts. In 1997, anthrax bacterium (Bacillus antracis) was modified by an introduction of an alien gene, making it resistant to existing vaccines. In 2012, two studies reported genetically modifying H5N1 avian flu virus (Morens, 2012; Nature, 2013), a flu variant with extremely high mortality rates (WHO, 2020). The new strain was reported to be transmitted through the air in ferrets, which are a common animal model intended to approximate transmission in humans. To obtain the common H5N1 avian flu virus, it was necessary to spend extended periods of time with infected birds. The publication of five mutations engineered to enhance transmissibility could serve a hostile agent planning to create and release a pathogen with pandemic potential.

In 2014, another flu variant, H7N1, was modified to allow respiratory transmission in ferrets (Dermody, 2014a). The decision by the Journal of Virology to publish this research has sparked a debate (Wain-Hobson, 2014; Dermody, 2014b) prompting the American Society for Microbiology Journals to publish details of its DURC review process (Casadevall, 2015). 

Gain of function research is not limited to human pathogens: In 2011, a study was published on modified entomopathogenic fungi M. anisopliae with engineered dihydroxynaphthalene (DHN) melanin biosynthetic genes (Tsang, 2011). This created a strain with enhanced anti-stress capacity and virulence, making the fungi both a more effective killer of the diamondback moth (Plutella xylostella) larvae, and more resistant to UV-B light and extreme temperature, improving its survivability. This is just one of many instances of genetic modification of plant or animal pathogens to increase their resistance and enhance their virulence (Butler, 1998; Jacobson, 2000; Liu, 2002; Wilson, 2009).

Because of the clear bioweapon potential of GOF research on ‘select agents’ (pathogens identified as a major risk to public health), a moratorium should be sought. Furthermore, GOF  results are almost certainly prohibited from circulation, diminishing the value of undertaking the research.

 

Biological Toxins and Gene Editing

 

Gene editing an organism to produce and deliver a biological toxin is another way to engineer a bioweapon. Although biological toxins are chemical molecules, they are commonly produced by plants, animals, and microorganisms like bacteria, fungi, or algae. Therefore, gene editing of these organisms is considered to be one of the ways to create a bioweapon. In particular, there are many organisms that produce neurotoxins – biological toxins that target the nervous system. There are a number of paralytic neurotoxins produced in living organisms like bacteria and algae (Giordano 2011; Giordano, 2017). 

Gene editing can be used to obtain 

• genetically modified organisms that have enhanced abilities for the production of biological toxins, 
• increased stability and resilience to environmental conditions, or even
•  given the ability to produce a biological toxin where none previously existed. 

The developments in genetic engineering technology, the digitization of genetic information obtained through scientific research, and its wide proliferation online make it easier than ever to obtain the necessary information and conduct the experiment to create a bioweapon through genetic modification. With technological advances far out-pacing regulations, it is likely that this will become even easier to do in the future.

 

In Vivo Genetic Modification

 

Current gene editing technology has its limitations: While it is easy to conduct gene editing in cells and tissues in vitro (lat. in glass; meaning in a glass dish), it is significantly harder to alter a genome in a living organism – in vivo, especially complex organisms like mammals. There are several challenges current technology faces when it comes to in vivo genetic modification.

CRISPR and other gene editing technologies use molecules as editing tools. In order to conduct genetic modification in vivo, these gene editing molecules must be injected into the organism (for humans, this would be either into the muscle tissue or blood). Once inside, the gene editing molecules would have to be viable inside the body and be able to cross cellular membranes to get to the cell DNA. In addition, for genetic modification of the nervous system and the brain, these molecules would have to be able to cross the blood-brain barrier. 

All of these conditions make viruses the most likely delivery system for in vivo genetic modification. Customized genetic viral vectors, with adeno-associated viruses (AAV)) have been successfully created for non-invasive gene transfer in mice (Deverman, 2016; Chan, 2017).  “In the adult mouse, intravenous administration of 1 × 1011 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1 × 1012 vg of AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons.” (Chan, 2017).

“However, this method (viral gene transfer) presents several side effects such as generation of novel infectious agents, immunogenicity of the vector and mutational insertion of viral DNA” ( Pasquet et al, 2018) 

The AAV technology is currently being widely used to develop gene therapies for a number of conditions, including coagulation disorders, inherited blindness, and neurodegenerative diseases (Colella, 2018; Wang, 2019). On 13 November 2018, there were 145 interventional clinical trials involving rAAV registered at ClinicalTrials.gov (Wang, 2019).

“AAV belongs to the genus Dependoparvovirus within the family Parvoviridae. Its life cycle is dependent on the presence of a helper virus, such as AdV (adenovirus), hence its name and taxonomy classification. AAV is found in multiple vertebrate species, including human and non-human primates (NHPs). The current consensus is that AAV does not cause any human diseases. It is composed of an icosahedral protein capsid of ~26 nm in diameter and a single-stranded DNA genome of ~4.7 kb.” (Wang, 2019)

AAVs are modified to replace their protein-coding genetic sequences with therapeutic gene expression cassettes, contributing to their low immunogenicity and cytotoxicity when delivered in vivo. Its single-stranded DNA is first converted to double-stranded DNA, by combining AAVs containing each of the halves of the double strand, which are then annealed by Watson–Crick base pairing in the nucleus. Another approach is to design the AAV to generate a self-complementary genome configuration, removing the need for two AAVs, but also halving the packaging capacity of the genetic cassette.

There are several different gene therapy strategies: gene replacement, gene silencing, gene addition, and gene editing (Wang, 2019). Gene therapy in adult animals targets a specific tissue, like the liver, eye, or muscle. Sometimes, transduced tissue can be used to produce therapeutic proteins for the treatment of disease in another tissue – for instance, transduced muscle tissue can be used to treat non-muscle disorders. Non-invasive gene transfer into cells or tissue by application of electric pulses, is also being tested. This could present risk of misuse in terms of remote gene transfer.

Any of these strategies can conceivably be employed to create a bioweapon: – 

• Transduced tissue can be coded to produce toxic proteins instead of therapeutic ones;
•  Genetically modified neurons can be coded to lose cognitive function., while 
• Liver and pancreas cells can be coded to alter their function and cause disease. 

There are many more possibilities for a human’s genome to be attacked to cause disease or death, and new technologies are exploring many of these avenues, albeit in a quest to cure or prevent disease. The knowledge necessary to achieve clinical success is not there yet: Even the majority of natural  microorganisms and their functions have not been studied yet. 

Regardless of risk, there has been rapid development in this consequential research and it is important that the risks are identified in advance.  

 

Synthetic Biology: Bioweapons Application

 

Synthetic biology has massive potential for exploitation in the development of bioweapons. It is used widely in the production of pathogens and corresponding vaccines.

The medical advances of the past century, vaccines in particular, have contained and almost eradicated some of the worst diseases known to man. Therefore, such pathogens cannot be found in nature, making it extremely difficult, in the past, for a malicious agent to obtain a sample of the pathogen to develop into a bioweapon. 

Now, though, synthetic biology and biotechnology allow for the re-creation of many dangerous pathogens without the need to obtain a live sample. All that is needed is the genetic code of the pathogens in digital form, which typically can be found in databases online.

In 2002, the first case of a synthesized pathogen was published for a poliovirus (Cello, 2002). The researchers used the digital genetic sequence of the virus, ordered small customized DNA sequences, and combined them to reconstruct the complete viral genome. Finally, they created a live virus by adding a set of chemical agents, and the virus started to  reproduce. Poliovirus has a relatively small and simple genome, with around 7500 nucleotides (7.5 kb). The synthesized pathogen would constitute an ineffective bioweapon because; 

• most of the world’s population is vaccinated, 
• the disease develops in around 0.5% of cases, and 
• it spreads through infected fecal matter or saliva. 

According to WHO, there were 147 cases of polio globally in 2018, and 539 in 2019, down from around 350,000 in 1988. All of the cases of wild polio were recorded in Afghanistan and Pakistan, showing how the disease has been contained in the rest of the world. Although the poliovirus would not be effective as a bioweapon, this experiment shows how an artificial virus can be synthesized relatively easily in a lab with just a digital viral genome. It was only a matter of time before a more dangerous pathogen would be synthesized.

This occurred in 2017, when a group of Canadian scientists, as part of an effort to create a safer smallpox vaccine, synthesized the horsepox virus. Using around $100,000 worth of chemicals, equipment and staff who were largely untrained in genetic engineering or synthetic biology, they applied the same method to recreate the related variola virus that causes smallpox (Noyce, 2018a). The close relation of the horsepox virus to the variola virus suggested that the same method could be applied to recreate the variola virus. Back in 2003, a national security report had already warned about just this eventuality when considering the national security implications of synthesizing the poliovirus (Van Aken, 2003):

“However, the method for creating polio virus artificially cannot be directly transferred to the smallpox virus. The variola genome, with more than 200,000 base pairs, is far bigger than that of polio, and even if it were possible to recreate the full smallpox sequence in vitro, it could not easily be transformed into a live infectious virus particle. But there might be other ways. It would, for example, be possible to start with a closely related virus, such as monkeypox or mousepox, and to alter specifically those bases and sequences that differ from human smallpox.”

In assessing the risk of Smallpox being used as a bioweapon, consider that it 

• Has been eradicated as a disease, 
• There have been no reports of the wild virus, 
• The last vaccinations occurred in 1980. 
• That means most of the world's population is not vaccinated against smallpox, 
• There are no large vaccine stocks at hand to conduct vaccination in case of an outbreak, a 
• The disease has a mortality rate of around 20% in adults and 80% in children under the age of 5. 
• The live virus exists only in two high-security labs in the US and Russia, posing minimal risk from accidental release, although there have been biosafety breaches in high security laboratories in recent years. Also Russia is one of five countries who maintain a complete bioweapons program, even having signed the BWC treaty. 

“At its peak, the Soviet program involved some 65,000 scientists, technicians, and other workers hidden in dozens of facilities operated by the KGB, the Soviet Academy of Sciences, the Soviet Academy of Medical Sciences, and the Ministries of Defense, Agriculture, Health, and Chemical Industry. Much of this illegal biological weapons program was hidden in plain sight in facilities conducting research and development (R&D) for pharmaceutical, industrial, and other civilian purposes.

Recent research projects indicate that the technological obstacles to the creation of an artificial variola virus are relatively small. Any number of national and non-national actors could successfully do it, giving them access to a live, infectious virus from the smallpox family. 

Whether it is intended for use as a bioweapon or not, the risk of accidental release or theft of the virus for a biological attack becomes significantly greater as the number of locations containing the live virus increases.

This has prompted a considerable debate about the risk evaluation of a potential smallpox epidemic (DiEuliis, 2017; Koblenz, 2017; Noyce, 2018b; MacIntyre, 2020). Prof. Koblenz of George Mason University wrote:

“The synthesis of horsepox virus takes the world one step closer to the reemergence of smallpox as a threat to global health security. That threat has been held at bay for the past 40 years by the extreme difficulty of obtaining variola virus and the availability of effective medical countermeasures. The techniques demonstrated by the synthesis of horsepox have the potential to erase both of these barriers. The primary risk posed by this research is that it will open the door to the routine and widespread synthesis of other orthopoxviruses, such as vaccinia, for use in research, public health, and medicine. The normalization and globalization of orthopoxvirus synthesis for these beneficial applications will create a cadre of laboratories and scientists that will also have the capability and expertise to create infectious variola virus from synthetic DNA. Unless the safeguards against the synthesis of variola virus are strengthened, the capability to reintroduce smallpox into the human population will be globally distributed and either loosely or completely unregulated, providing the foundation for a disgruntled or radicalized scientist, sophisticated terrorist group, unscrupulous company, or rogue state to recreate one of humanity's most feared microbial enemies. The reemergence of smallpox—because of a laboratory accident or an intentional release—would be a global health disaster.” (Koblenz, 2017)

In response to the criticism, the researchers who synthesized the horsepox virus made several points (Noyce, 2018b): 

• “All attempts to oppose technological advances have failed over centuries”,
•  “One should instead focus on regulating the products of these technologies”
• “Educate people of the need to plan mitigating strategies based upon a sound understanding of the risks.” 

The National Science Advisory Board for Biodefense (NSABB) eventually agreed that their oversight framework should be applied uniformly beyond the life sciences’ federally funded research, to synthetic biology, academia and the private sector. But not to classified biodefense analysis and countermeasures - which includes Research of  Concern.

The creators of the horsepox also added that pathogens like the variola virus are already under strict regulatory control in many countries: 

“Possession of variola virus is a crime in Canada, and other countries have similar laws. Because there are DNA clone libraries, WHO recommends that no one should own >20% of the variola genome outside of the two authorized sites. Many countries follow these policies, and some legislate greater restrictions on the size of cloned variola sequences. Therefore, from a biosafety and biosecurity perspective, we already have some controls in place to manage the products of these technologies.

Most of the regulatory recommendations are voluntary and follow vague criteria, rather than mandatory screening and enforcement.

• Finally, they concluded that many technological advances that provided great benefit to humanity were initially seen as a threat, and that includes genetic engineering, adding:

“As the memory of smallpox and polio fades, the challenge will be to educate new generations about the risk posed by these diseases. This necessitates providing the ongoing support that public health agencies will need to protect populations from even “extinct” epidemic diseases (emphasis added). The advance of technology means that no disease-causing organism can forever be eradicated.”

This sort of rhetoric plays neatly into the hands of the pharmaceutical industry to justify continuing to recreate high risk deadly diseases and produce vaccines for them. 

The reality of technological developments that allow the synthesis of pathogens that have been eradicated poses a unique national security threat.. World governments are required to step up and agree that the risks of conducting this research are too high and implement either a ban or regulatory framework capable of enforcing existing legislation. We might otherwise be faced with an epidemic of a disease that has been long forgotten or a new synthetic strain. 

The other important issue is the risk evaluation and justification of such work: the main purpose of global vaccination programs is to eradicate diseases so the vaccination would no longer be necessary. The creation of artificial pathogens defeats that purpose completely, creating an endless need for vaccination against terrible diseases simply because there is a risk from a release of an artificial pathogen. One must wonder whether this research and the knowledge obtained are worth imposing this high cost on the entire world. 

Multiplex Automated Genome Engineering (MAGE)

 

One of the main disadvantages of synthetic biology is the lack of genetic diversity of its products. Genomic diversity is the most important feature of natural organisms, allowing populations in nature to adapt to a variety of environments. This diversity is a byproduct of the long natural evolution and selection processes that occur in the wild. This is extremely difficult to reproduce in laboratory conditions, as the evolutionary processes occur over a relatively long time (Elena, 2003) - 3.7 billion years of continuous, undisturbed evolution by natural selection, to be exact .

To address the lack of diversity, a method was developed - multiplex automated genome engineering (MAGE), which accelerated evolutionary processes in the lab (Wang, 2009):

“…we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate the rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions).”

The researchers applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce lycopene (Wang, 2009). They simultaneously modified 24 genetic components using a pool of synthetic DNA, creating 4.3 billion genomic variants per day. Within three days, they isolated variants with increased lycopene production.

The MAGE method creates strain libraries for large-scale genotype–phenotype mapping (Si, 2017). The process of genetic engineering typically requires both the identification of genetic determinants and optimization of their expression in a concerted manner to improve target traits. Most of the methods perform these two tasks separately by “either modifying individual genes on a genome scale or creating combinatorial diversity among pre-defined targets” (Si, 2017). Automation of multiplex genome-scale modifications provides both, at the same time; genetic diversity and genome-wide screening and multiplex optimization to identify and isolate strains with improved traits. 

These recent advances – the development of MAGE method and its automation, provide the tools that allow rapid genome-scale engineering that can produce multiple simultaneous changes to the genome and then use accelerated evolution to screen for and isolate strains with desired traits. The rapid mutation and evolution process prevents the observation of, let alone study or biological correction of mutations, including those that are harmful or not beneficial. 

MAGE changes the paradigm of genetic engineering: Instead of sequential editing of genomic targets where the desired editing event typically occurs at a very low frequency, MAGE can perform simultaneous editing of multiple targets and screen an enormous number of strains for desired gene editing events (Bao, 2016). 

Some  of the national security implications from MAGE technology are:

• Automated facilities are vulnerable to remote operation, release and theft.
• CRISPR, including the integration of synthetic DNA into existing organisms are produced with no risk assessment and unspecified protocols on waste management.. 
• The automated process allows the rapid creation and screening of billions of novel micro-organisms every day
• No countermeasures or treatments exist. 
• Screening different strains to identify those which underwent desired gene editing events and isolate those with desired traits. Indeterminate methods to identify and manage those that didn’t
• Operators of MAGE laboratories should be assigned liability and responsibility for biosafety. 
• Projects are not required to provide evidence of safety testing
• Facilities do not have to produce an inventory to prove they are operating within the legal limit for storage, transfer and other Biosafety guidelines.
• The automated process allows the rapid creation and screening of billions of novel microorganisms every day, sufficient to serve as a bioweapon.

 

Conclusion

 

Recent rapid advances in biotechnology have opened up new possibilities for bioweapons development. Biological weapons were the first weapons of mass destruction to be identified and made illegal worldwide. Unlike the use of nuclear weapons which is decided at a national level, anyone can launch a bioweapon. 

Gene editing technologies have made biological weapons accessible to private national and international interests. The weaponization of biological agents is now affordable and potentially profitable. The large scale creation of engineered microorganisms and virus vectors can easily produce pathogens that:

Render a vaccine ineffective;

Confer resistance to antibiotic or antiviral agents;

Enhance the virulence of a pathogen or render a non-pathogen virulent;

Increase the transmissibility of a pathogen;

Alter the host range of a pathogen;

Enable evasion of diagnosis or detection methods; and

Enable weaponization of a biological agent or toxin.

(Paquet, 2018)

It is easier, more accessible and more affordable than ever before for small factions to engage in bioweapon development. The threshold of technical and scientific knowledge and capacity has been lowered significantly by the new technologies. This allows people with little to no specialized training to engage in genetic engineering. More importantly few scientists and none of the automated machines in MAGE facilities are screening for harmful mutations. 

When the online ‘Library’ of genetic sequences is described, it is not clear if there is a category for Potential Biological Weapon or Potential Pandemic Pathogen or Special Agent - which is another term for a biological agent that poses a pandemic risk to public health.

In response to the serious national and global security implications, governments should urgently re-enter protocol negotiations on how to enforce the Biological Weapons Convention legislation. The current pandemic should provide additional leverage for regulatory bodies to demand more than voluntary reporting on biosafety, biosecurity, evidence of the benefits of the research and how the information can be safely shared. 

 

If proposed health benefits are being used to justify genetic research and the taxpayers’ investment, but the results being stopped from publication, then this serves the pharmaceutical industry too; in keeping their research and patented products out of regulatory scrutiny.

Since the collapse of negotiations as to how to implement the Biological Weapons Convention’s governance,, the onus has been on scientists and individual research programs to follow voluntary Codes of Conduct. WHO stated that scientists themselves were in the best position to assess the risk of their own work. The priority given by the World Health Organization was to raise awareness of biosafety risks among life scientists through training and education. They hoped a culture of responsibility would facilitate this process.

 

CRISPR has made the creation of biological toxins and their genetic delivery straight forward. Multiplex Automated Genome Engineering (MAGE) has made it possible to scale up production, simultaneously edit multiple targets, with no human scientific or ethical oversight. In vivo gene editing has become a reality, with hundreds of clinical trials being conducted in the US alone. Synthetic biology with its low genetic diversity is being widely used, with no impact studies on the multiple functions of existing microbiomes in human and environmental health. Any of the products of these technologies could be utilized to harm even more readily than perform their stated objectives, such as cure disease. 

Gain of function research is explicitly used to produce more virulent strains of known diseases with pandemic potential pathogens, while synthetic biology has been shown to have the potential to create artificial pathogens to recreate any disease.

 It might be tempting to conclude that the risks are exacerbated by the wide availability of information online: Genome databases that contain digital genome information on pandemic pathogens. However, it is relevant to look at the threat of bioweapons from other perspectives, including motivations to create a bioweapon; what might be the objective of an attack and who is funding the research. 

For dual use technology research alone, 7.6 billion dollars has been spent; 6.1 billion by taxpayers and 1.5 billion by the military (Kennedy, 2021) Military funding could point to these technologies specifically being developed for military purposes, including biological weapons and surveillance. This would be another reason to withhold the results of the research, as classified. Already the National Defense Strategy research is exempt from oversight, even though it evolved from the US Bioweapons Program. It now focuses on strategies to assess, prevent, protect against, respond to, and recover from biological threats. They have an exemplary R&D group DARPA who lead the way in coupling innovation with biosecurity. DARPA started the Safe Gene program in 2018 to develop safety guidelines including: 

Techniques that allow gene editors to be switched off and back on again once inside a person or organism; 

Development of drugs and other agents to block or reduce the action of gene editors; and 

Tools to clean up environmental genomic “spills” and leave things as they were before 

Developing and validating methods of molecular confinement that minimize the risk of unwanted genome editing. 

https://www.phe.gov/Preparedness/biodefense-strategy/Pages/default.aspx (at bottom as well)

New assessments of gene-editing, synthetic biology and gain of function research, in the context of bioweapons, may be needed. However, the unreasonable risks are both obvious and comparable, whether  the release is intentional or accidental. 

Noyce and Evans warned that the “advance of technology means that no disease-causing organism can forever be eradicated.” This fundamentally changes the paradigm of the global health strategy, which is predicated on the ability to eradicate diseases, using vaccination or other immunization tools. Once eradicated, a disease like smallpox would never be a threat to our communities and the economy. 

However, modern biotechnology allows a wide array of agents to resurrect deadly viruses and reignite diseases that have already been eradicated at a great cost. This raises the question about the proper balance of interests: the global community and humanity on one side, and a small group of scientists,  biotechnology companies, pharmaceutical and military interests on the other. The former would no doubt strongly prefer these eradicated diseases to remain extinct, and see a ban on dangerous research. The latter see an interest in pursuing this avenue of research, without consideration for the broader public interest.

Therefore, the role of governments becomes more important than ever: Tto monitor, regulate, and prevent any research that poses an unacceptable risk to the public. Academic institutions must develop young scientists’ awareness of the major threat of gene edited microorganisms entering the human or wider gene pool. Training should embed a code of conduct that takes into account the unpredictable interaction of genetically engineered organisms in nature and the potential misuse and weaponization of the technologies and their research. 

Our society has made great advances in the past several decades and managed to suppress, contain, or eradicate many infectious diseases that have plagued humanity for millennia. However biotechnology companies have also managed to suppress, contain and eradicate many scientific studies which question the safety of their products and the genetic engineering process itself. That research could have prevented the emerging and established conditions associated with GMOs in our food supply.  It is irresponsible for genetic research to be allowed to proliferate potential bioweapons; having not had to defend their reason for conducting the research in the first place; being under no obligation to share results and having provided no Biosecurity Evaluation. The legally-binding Biological Weapons Convention has been difficult to enforce until now. Each research project could be required to send an evidenced response to these basic guidelines, to get a license to proceed:

• Preventing the development and possession of biological warfare agents or weapons; 
• Controlling access to dual-use biological materials, equipment, or associated information that could be used for hostile purposes; 
• Promoting the safe and secure handling of pathogens and toxins inside and outside the laboratory; 
• and ensuring that the risks from the most consequential types of biological research are properly identified, assessed, and mitigated before the work is carried out.

 

Accepting that the threat from genetically engineered microbes is one of ‘mass destruction’ it is irresponsible to value unrestricted use of technology and commercial interests above it.

References

 

Bao, Z., Cobb, R.E. and Zhao, H., 2016. Accelerated genome engineering through multiplexing. Wiley Interdisciplinary Reviews: Systems Biology and Medicine, 8(1), pp.5-21.

Butler M. J., Day A. W.. 1998. Fungal melanins: a review. Can. J. Microbiol. 44:1115–1136

Casadevall, A., Dermody, T.S., Imperiale, M.J., Sandri-Goldin, R.M. and Shenk, T., 2015. Dual-use research of concern (DURC) review at American Society for Microbiology Journals. (Available at: https://mbio.asm.org/content/mbio/6/4/e01236-15.full.pdf)

Chan, K.Y., Jang, M.J., Yoo, B.B., Greenbaum, A., Ravi, N., Wu, W.L., Sánchez-Guardado, L., Lois, C., Mazmanian, S.K., Deverman, B.E. and Gradinaru, V., 2017. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nature neuroscience, 20(8), pp.1172-1179.

Cello, J., Paul, A.V. and Wimmer, E., 2002. Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science, 297(5583), pp.1016-1018.

Colella, P., Ronzitti, G. and Mingozzi, F., 2018. Emerging issues in AAV-mediated in vivo gene therapy. Molecular Therapy-Methods & Clinical Development, 8, pp.87-104.

Dermody TS, Sandri-Goldin RM, Shenk T. 2014a. Sequence changes associated with respiratory transmission of H7N1 influenza virus in mammals. J Virol 88:6533–6534. http://dx.doi.org/10.1128/JVI.00886-14.

Dermody TS, Casadevall A, Imperiale MJ, Sandri-Goldin RM, Shenk T. 2014b. The decision to publish an avian H7N1 influenza virus gain-of-function experiment. mBio 5:e01985-14.

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