Dual Use Biotechnology Notes

Modified Agents of Biological Origin

• Modified agents of biological origin are potential biological weapons.

Presentation Information

• This presentation was created by Dr. Magdalena Florek from the Wrocław University of Environmental and Life Sciences.
• It is intended for:
  • ED students of the III year of the Faculty of Veterinary Medicine in Wrocław.
  • Erasmus students of the Faculty.
• The presentation is for teaching purposes only.
• Further dissemination, recording, copying, or making changes is prohibited due to copyright protection.

Biotechnology Definition

• According to the Convention on Biological Diversity, biotechnology is defined as: “any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use.”
• Frequently used organisms or their derivatives include: micro-organisms, animals, plants (or their cells), and enzymes.
• These can be used to process substances, renewable materials, or serve as sources for valuable substances or goods.

'Dual Use' Concept

• In arms control and disarmament, 'dual use' refers to technologies intended for civilian application that can also be used for military purposes.
• The dual-use research dilemma in the life sciences refers to the problem of producing and publishing research within the life sciences that is directed toward or intended to improve public health, animal health, or agricultural productivity, but that in the hands of a terrorist could be used to impair public health.
• Biotechnology advancements enable progress in medicine (e.g., antimalarial drugs, rapid flu vaccine synthesis) but can be misused.
• Weaponization or mismanagement of modified biological agents can lead to significant loss of life, as well as ecological and agricultural damage.

Challenges due to Rapid Developments in Biotechnology

• Rapid developments in biotechnology, genetics, and genomics are creating a variety of environmental, ethical, political, and social challenges (necessitating regulations).
• The expansion of modern biotechnology in medical and pharmaceutical research and production has led to a worldwide availability of knowledge.
• The rapid development of molecular biology and metabolic engineering tools has enabled the development of chimeric organisms with non-native characteristics.
• Biomedical research developments open up avenues for the creation of new biological weapons.
• Nearly all countries have the technological potential to produce large amounts of pathogenic microorganisms.

Concerns Associated with ‘Dual Use’ in Biological Sciences

• Availability of numerous culture collections provide bacterial, fungal, or viral strains. The World Federation for Culture Collections lists 581 Culture Collections in 68 countries, holding over 1.6 million culture samples.
• Information regarding naturally-occurring organisms and gene sequences is freely accessible. Genetic information is available at www.ncbi.nih.gov/Genbank/ and other bioinformatics repositories.
• The Internet provides free access to bioinformatics applications and software tools for analyzing genomic information.
• Knowledge links nucleic acid sequence information with the biological functions of proteins.
• Commercial DNA sequencing companies provide a baseline capability for genetic engineering of microorganisms.
• Technical questions can be answered at help sites or message boards (e.g., Research Gate).

Global Knowledge Base Expansion

• As the global knowledge base expands and is dispersed, generating genetic combinations that increase infectivity and pathogenicity of organisms becomes easier and inexpensive.
• Required materials (e.g., reagents, culture media, host vectors) are readily available worldwide.
• Examples of easy access or procurement include the International Genetically Engineered Machine Competition (iGEM) and the do-it-yourself biology (DIY bio) movement.
• Synthetic biology is increasingly accessible and affordable across organizational structures.

Examples of Scientific Research with Potential for 'Second Use'

• Genetic engineering could lead to the development of new, more effective biological weapons.
• Introduction of antibiotic resistance into bacterial pathogens.
• In the former Soviet Union, a gene of $B. cereus$ was introduced into $B. anthracis$, altering its immunological properties, making existing vaccines ineffective.
• In the US, the US Naval Research Laboratory developed genetically engineered fungi that degrade materials like plastics, rubber, and metals.
• In the 1990s, the US studied fungi to kill drug-producing crops: $Pleospora papaveracea$ (against opium poppy, tested in Tashkent, Uzbekistan) and $Fusarium oxysporum$ (developed to kill coca plants, project halted due to international protests).

’Dual Use’ - COVID-19

• An article exemplifies the complicated idea of ’dual use’:
• Scientists reconstructed the SARS-CoV-2 rapidly using a synthetic genomics platform (yeast based), in order to accelerate therapeutics and vaccine development.
• Given the dual-use nature of this technology, there exists a high biosecurity risk.
• The two basic values of safety/security and intellectual freedom of research must be considered evenly.
• This paper calls for a careful assessment to the risks of the technology, replacing risky technologies with safe ones.
• The risks of publication also need to be strictly assessed.
• The authors believe that in addition to enhancing the “self-government” and self-discipline of scientists and scientific communities, government supervision must be reinforced, laws and regulations should be improved, and a global regulation framework ought to be constructed.

'Dual use' - COVID-19 (cont.)

• A research group published a non-peer-reviewed paper titled "Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform" on BioRxiv.
• The authors claimed to be able to engineer and resurrect chemically-synthesized clones of SARS-CoV-2 with a yeast-based synthetic genomics platform.
• This paper presents the genetic reconstruction of diverse RNA viruses, including the novel coronavirus.
• The authors believe that by using this platform, generation of SARS-CoV-2 from chemically synthesized DNA could bypass the limited availability of virus isolates to allow genetic modifications and functional characterization of individual genes, as well as to generate serological diagnostics, to develop and assess antivirals and vaccines, and to establish appropriate in vivo models.
• During this critical period of global combat with this epidemic disease, the present research could contribute to the development of antiviral therapeutics and that of a vaccine.

Risks Related to COVID-19 Research (Dual Use)

• SARS-CoV-2, a virus with high transmissibility and susceptibility, poses a biosecurity risk wherein bioterrorists could exploit this characteristic, with potentially hazardous consequences.
• Publishing the technology roadmap enables scientists and terrorists to synthesize more complex viruses or develop a "super virus" with extremely high infectivity, virulence, or vaccine-resistance.
• The internet facilitates ordering biological materials for synthesizing bioweapons.
• Accidental leakage of synthesized virus particles from the laboratory increases biosafety risks, threatening the safety of humans as well as that of the ecological environment.
• There exist two concepts regarding the autonomy or regulations:
  • Weaker autonomy, Stronger regulation, Security (goverment institutions)
  • Stonger autonomy, Weaker regulation, Progress of science (individual scientist or research institution)

Genetically Modified Organisms (GMO)

• A genetically modified organism is one whose genetic characteristics have been altered by the insertion of a modified gene or a gene from another organism using the techniques of genetic engineering.
• Genetically modified organisms encompass a wide spectrum of single and multicellular organisms, including plants and animals.
• This effort specifically addresses microorganisms.
• Organisms modified by insertion of genes from another organism are also referred to as “transgenic” organisms.

(Adverse) effects of GMO use

• Potential adverse effects of GMOs vary and may include:
  • Disease in humans (including allergenic or toxic effects).
  • Disease in animals and plants (including toxic, and in some cases, allergenic effects).
  • Effects on the dynamics of populations of species in the receiving environment and the genetic diversity of each of these populations.
  • Altered susceptibility to pathogens facilitating the dissemination of infectious diseases and/or creating new reservoirs or vectors.
  • Compromising prophylactic or therapeutic medical, veterinary, or plant protection treatments, for example by transfer of genes conferring resistance to antibiotics used in human or veterinary medicine.
  • Effects on biogeochemistry (biogeochemical cycles), particularly carbon and nitrogen recycling through changes in soil decomposition of organic material.

Objectives of Modifying or Creating a Threat GMO

• Increase of infectivity.
• Increase of virulence/mortality rate.
• Diminish host immunity or confer antibiotic resistance.
• Increase of survivability outside the host (i.e., environmental stability).

Methods to Modify or Create a Threat GMO

• Introduction of genes into an existing natural organism or by completely artificial “abiotic” synthesis of organisms.
• Modification of the organism by putting a molecule (targeting certain host’s receptors) on the surface of an existing pathogen, that causes it to bind more efficiently to the host target cells (to increase infectivity).
• Insertion of a genetic material (e.g., in a form of plasmid) that encodes for a toxin, resistance, virulence factors, etc.
• Insertion of the DNA sequences resulting in host’s immunosuppression.

'Dual Use' of Research/GMO as a Potential Threat

• Recent developments bearing on the potential threat of genetically modified or synthetically produced microorganisms:
  • In 1981, scientists cloned a full-length Poliovirus genome that was infectious to mammalian cells and demonstrated the basis of the ability to replicate an infectious RNA virus. Then, in 2002, purely chemical synthesis of an infectious Poliovirus in the absence of any natural template has been reported.
  • In recent work, DNA sequences on the order of 1 million base-pairs have been synthesized entirely from digitized genome sequence information, and the resulting organisms were phenotypical and capable of self-replication.

'Dual Use' of Research/GMO as a Potential Threat (Cont.)

• The potential for modifying organisms to significantly enhance virulence and mortality rate (example of gain of function studies), was shown when modified mouse pox virus, intended for use as a contraceptive, proved 100% deadly to mice (even previously vaccinated).
• The complete genome sequence of 45 variola strains providing supplemental material with gene organization of the virus has been published.
• Cloning and recovery of infectious Ebola virus and of a mutant more cytotoxic than the, natural wild-type, has been reported.
• In 2008, Israeli researchers published a procedure for the de novo construction of error free DNA molecules from error-prone commercially available oligonucleotides. This ability was cited as having the potential to allow masking of an intended synthetic molecule or organism during purchase of oligonucleotides.

The Development of Biological Weapons

• The development of effective biological weapons requires an intense research program to solve complex problems:
  • The production of virulent strains of suitable agents.
  • The mass production of the agents without loss of pathogenicity.
  • The development of an effective means of delivery (this is very demanding and has rarely been accomplished to date).
• Access to highly virulent pathogens that are necessary for the process, is strictly regulated, but artificial synthesis of agents or new combinations of agents becomes more possible (e.g., artificial polio virus has been synthesised de novo using the genetic sequences, which are available online).
• The mentioned method could be used to synthesize other viruses with similarly short DNA sequences (e.g., Ebolavirus, Marburgvirus and Venezuelan equine encephalitis virus).

The Development of Biological Weapons (Cont.)

• Viruses like Variola major are more complicated but with time, it should become possible to create them in a laboratory (several genes of the virus has already been reconstructed artificially, in South Africa).
• Almost twenty years ago, researchers documented for the first time that the sequence of a pathogenicity-related gene from the vaccinia virus could be transformed through the targeted mutation of 13 base pairs into the sequence of the corresponding smallpox gene, so related viruses of animal-associated pox (vaccinia, cow pox, monkey pox (!!!!) , etc.) may be used to create human pathogen.
• Considering the extreme danger that smallpox poses to a population, it seems questionable to make the smallpox sequence publically available (another example or ‘dual use’ idea).
• New types of biological weapons are becoming possible to create, like so-called ‘non- lethal’ weapons (e.g., that are targeted against materials).

The JASON Scientific Group Classification

• In 1997, a study was conducted by The JASON scientific group in order to identify future threats and uses of advanced biological warfare agents. Six classes of genetically engineered pathogens that could pose serious threats to society were indicated.
  • Binary biological weapon: this is made up of a two-component system with independent elements that are safe to handle separately but when mixed together form a lethal combination (e.g., virus and helper virus, or bacterium and bacterial virulence plasmid). The process of generating this type of pathogens has been decoded as revealed by a former Soviet Union defector („super plague” - antibiotic resistant, shifting virulence - both plasmid dependent).
  • Designer genes (and life forms): using available knowledge/databases; choice of certain genes in order to create new biological life forms, presenting the most lethal characteristic. Those interested in developing a weapons can openly use the genomic sequence databases to choose the genes they want to use (e.g., involved in pathogenicity and virulence, adhesion and colonization of host cells, immune-response evasion and antibiotic resistance).

The JASON Scientific Group Classification (Cont.)

• Gene Therapy as a Weapon: replacement of a problematic gene with a good one, to normalize the condition of the recipient. It requires a vector - commonly genetically altered viruses (retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex) are used. Mutated genes (used as BW) can be delivered using this method.
• Stealth Viruses: tightly regulated, cryptic viral infections that can enter and spread in humans cells using vectors, stay dormant for a period of time, and then are triggered by an internal or external signal (e.g., latency reversion or carcinogenic transformation).
• Host Swapping Diseases: viruses causing asymptomatic infections in their hosts may be engineered in order to be transferred into new host species, in which severe manifestations of the infection may occur.
• Designer Diseases: conceptually design disease transferred by newly created/altered pathogen. It might work by attacking the immune system or it might reactivate dormant genes to cause transformation of cells (e.g., spread of cancer), or induce apoptosis.

GoF (gain-of-function) Experiments

• Experiments in which new phenotypes (properties) are added to pathogens.
• Any selection process involving an alteration of genotypes and their resulting phenotypes is considered a type of GoF.
• Research microbiologists use gain- and loss-of-function experiments to understand the genetic makeup of microbes and the specifics of pathogen-host interaction. GoF research often involves studies that enhance the pathogenicity, transmissibility, or host range of a pathogen to better understand the threat.
• GoF experiments can be highly beneficial (e.g., generation of pest resistant crops, microbes expressing proteins as recombinant human insulin, new cancer therapies enhancing lymphocyte function).
• However, GoF studies could be an issue of "dual-use" concern - the threat of accidental lab release or the pathogens becoming bioterror threats if they fall into the wrong hands.

The Categories of GoF Studies Performed on Viruses

• The first category (“gain of function research of concern,” group) includes the generation of viruses with properties that do not exist in nature (e.g., production of H5N1 influenza A viruses that are airborne-transmissible among ferrets, compared to the non-airborne transmissible wild type)
• The second category - the generation of viruses that may be more pathogenic and/or transmissible than the wild type viruses but are still comparable to or less problematic than those existing in nature
• The third category - in between the two first categories, includes the generation of highly pathogenic and/or transmissible viruses in animal models that do not appear to be a major public health concern

Biological Accidents

• Biological accidents occur regularly in laboratories
• When potentially infectious agents are involved, the effects are typically limited to the researchers present at the time and possibly their close contacts (but the pathogens usually are known to the medical community)
• Laboratory-acquired infections with pathogens that are particularly transmissible (such as influenza or SARS), could have consequences that go well beyond the laboratory
• If the pathogen is transmissible and novel, so that there is no immunity in the population, such an accident could become a global disaster (SARS-CoV-2?)
• The accidental release of a biological agent with synthetic components could also harm the environment
• Unsafe practices - performing work on contagious, modified pathogens in inappropriately low levels of containment or/and improper training - these could result in a breach of containment

Not Only Microorganisms

• The production of transgenic animals and plants that contain genetic elements from foreign sources and possess novel traits and characteristics also result in the creation of genetically modified organisms (GMO s) that can be potentially harmful to the environment and human health
• Potential health risks to humans include the possibility of exposure to new allergens, toxins, bioactive substances in genetically modified foods, as well as the transfer of antibiotic-resistant genes to gut flora