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Biodefense in the Age of Synthetic Biology (2018)

Chapter: 4 Assessment of Concerns Related to Pathogens

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Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
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Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
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Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
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Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
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Page 50
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
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Page 51
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 52
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 53
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 54
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 55
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 56
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 57
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 58
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 59
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 60
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 61
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 62
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 63
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 64
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 65
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 66
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 67
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 68
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 69
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 70
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 71
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 72
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 73
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
×
Page 74
Suggested Citation:"4 Assessment of Concerns Related to Pathogens." National Academies of Sciences, Engineering, and Medicine. 2018. Biodefense in the Age of Synthetic Biology. Washington, DC: The National Academies Press. doi: 10.17226/24890.
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Page 75

Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

4 Assessment of Concerns Related to Pathogens The use of disease as a weapon is thought to date back to at least the Middle Ages, when the Tartars used catapults to hurl plague victims over protective walls in the city of Caffa (Wheelis, 2002). Settlers to North America presented Native Americans with blankets that had covered smallpox victims, potentially exposing this naïve population to the scourge of smallpox (Duffy, 1951). With the advent of microbiological techniques, it became possible to use specific pathogens as weapons. This capability enabled several nations, but most extensively the Soviet Union and the United States, to develop offensive biological weapons programs, which continued until they were legally prohibited by the Convention on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on their Destruction (known as the Biological Weapons Convention, or BWC), signed in 1972 (BWC, 1972). After the BWC was signed, the development of pathogens as weapons became the province of clandestine nation-state programs and non-state actor terrorism. One of the most high-profile uses of pathogens as weapons was the “Amerithrax” bioterror attack in 2001, in which Bacillus anthracis spores were sent through the U.S. Postal Service, resulting in five deaths, prophylaxis of 30,000 individuals due to potential exposures, and hundreds of millions of dollars in decontamination expenses (DOJ, 2010). In these historical examples, naturally occurring pathogens were developed as biological weapons. Specific pathogens were selected for bioweapons development based on their ability to cause morbidity and mortality and on their ability to be converted into large-scale weapons. The age of synthetic biology raises the possibility that pathogenic bioweapons could be designed, developed, and deployed in new ways that depart from the disease-causing characteristics of a naturally occurring pathogen. First, while security protocols such as the Federal Select Agent Program and the Australia Group list (CDC/APHIS, 2017; The Australia Group, 2007) primarily in North America and Western Europe, have attempted to limit access to dangerous pathogens for many years, synthetic biology makes it is possible to synthesize genomes and use those to generate, or “boot,” copies of naturally occurring organisms in the laboratory, opening new opportunities for the acquisition of existing, regulated pathogens. Second, synthetic biology techniques could be used to modify existing organisms that are not subject to limited access regulations, potentially leading to the acquisition of desired attributes. For example, such manipulations could potentially result in pathogens that have, in comparison to the original pathogen, increased virulence, the ability to produce toxins or chemicals or biochemicals, antibiotic resistance, or the ability to evade known prophylactic or therapeutic modalities. Third, synthetic biology tools could be used to synthesize and boot entirely new organisms, potentially incorporating genetic material from multiple existing organisms (Zhang et al., 2016). This chapter analyzes these potential applications of synthetic biology related to the creation of pathogen-based bioweapons. To assess the level of concern warranted for each capability presented in this chapter (as well as those presented in chapters 5 and 6), the factors outlined in 47 PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 48 the report’s framework for assessing vulnerabilities were considered: Usability of the Technology, Usability as a Weapon, Requirements of Actors, and Potential for Mitigation. Conclusions regarding the relative level of concern for each capability as it relates to each factor are presented in the form of a five-point scale from low concern to high concern. Although all of the factors and elements identified in the framework were considered during the assessment, the discussion presented in these chapters focuses primarily on those elements deemed most salient to, or in some cases unique to, each capability. For each factor, the level of concern warranted for each capability relative to the other capabilities considered is presented at the end of the chapter along with a summary of the elements driving that relative level of concern. Conclusions regarding the relative ranking of all synthetic biology capabilities considered in the report are presented in chapter 9. RE-CREATING KNOWN PATHOGENS The construction of an organism from scratch requires at least two steps: synthesis of the organism’s genome and conversion of that nucleic acid into a viable organism (“booting”). Figure 4-1 illustrates these conceptual steps. This study assessed the potential for actors to use synthetic biology technologies to construct known, naturally occurring pathogenic organisms from scratch. The potential synthesis of viruses and bacteria are assessed separately because of their distinct biological features. At present, construction of eukaryotic pathogens with larger genomes—such as fungi, yeast, and parasites—is considered significantly more difficult and successes have not yet been reported. PREPUBLICATION COPY: UNCORRECTED PROOFS

49 Biodefense in the Age of Synthetic Biology FIGURE 4-1 Activities involved in the construction of an organism from scratch. NOTES: Considerations in the Design stage may include whether an exact copy of a pathogen sequence is desired, if synonymous mutations are introduced, or if a library (quasispecies) of sequences will be designed. Obtaining physical material in the Build stage may occur in the same physical location as the Design stage or may be outsourced to a commercial DNA synthesis provider. The size of the target sequence may make assembly necessary. Function of the synthesized pathogen, which may include the ability to infect and/or replicate, is determined in the Test stage. Re-Creating Known Pathogenic Viruses Using today’s technology, the genome of almost any mammalian virus can be synthesized, and the sequences of known human viruses are readily available through public databases such as GenBank®, an annotated collection of all publicly available whole and partial DNA sequences (NCBI, 2017). The 2002 synthesis of poliovirus by Eckard Wimmer and colleagues was among the first reported syntheses of a viral genome (Wimmer, 2006). The team assembled a complementary DNA (cDNA) of the poliovirus genome (approximately 7,500 nucleotides), under the control of the phage T7 promoter, from a series of oligonucleotides with an average size of 69 bases. This cDNA was used to produce viral RNA, which was then used to program an in vitro extract to produce infectious poliovirus virions (Cello et al., 2002). Since then, larger and PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 50 larger viral genomes have been generated, taking advantage of advances in the ability to synthesize longer and longer segments of DNA. Modern assembly methods have greatly expanded the scale at which DNA can be constructed, to the point that building the genome of virtually any virus—either in the form of the genome itself for a DNA virus or as a cDNA of an RNA virus that can be transcribed into the viral genome—is now possible (Wimmer et al., 2009). A notable example is the recent report of the construction of the horsepox genome (consisting of more than 200,000 base pairs) as part of an effort to develop a new smallpox vaccine (Kupferschmidt, 2017; Noyce et al., 2018). (It should be noted that while the booting of some viruses (e.g., polio) has been performed using cell-free extracts, most viruses must be booted inside cells, and some viruses, including horsepox, require the use of a helper virus in cells.) The assessment of concerns related to re-creating known pathogenic viruses is summarized here and described in detail below. Usability of Potential Usability as a Requirements the for Weapon of Actors Technology Mitigation Level of concern for Medium- Medium- High Medium re-creating known high low pathogenic viruses Usability of the Technology (High Concern) Overall, the cost of producing a viral sequence and booting it is fairly low; synthesis is inexpensive and becoming more so as time passes, and cell culture facilities are not expensive to build, maintain, and operate. Therefore, since the usability of the technology is hindered only by weak barriers, the level of concern with regard to this factor is relatively high. The Design phase of the Design-Build-Test cycle could be skipped for the synthesis of a known virus, assuming that the sequence of the genome to encode the pathogen is known. The first step of the Build phase would be to synthesize the DNA encoding the virus genome, which can either be ordered from commercial vendors or, if the actor has appropriate resources, synthesized in house. The former approach may present a barrier as most nucleic acid synthesis companies screen for sequences of concern, such as sequences derived from pathogens on the Federal Select Agent Program Select Agents and Toxins list (CDC/APHIS 2017). However, this barrier is weak for several reasons, including that actors need not limit themselves to viruses on the Select Agents list, industry compliance with the screening guidelines is voluntary, and oligonucleotide orders are not screened. Actors could exploit these factors or use other approaches to bypass screening, at least for viruses with smaller genomes. Having a genome in hand is only the first step in booting a viable organism. The ease with which a virus can be generated from its genome is largely a function of two variables: the size of the genome, and the nature of the genomic nucleic acid (i.e., DNA, positive-strand RNA, or negative-strand RNA). In general, the genome must be introduced into cells in culture in which the viral genome can be replicated and assembled into infectious viral progeny. If there is no cell PREPUBLICATION COPY: UNCORRECTED PROOFS

51 Biodefense in the Age of Synthetic Biology line in which the virus can be grown, the options become more limited. Poliovirus has been assembled completely in vitro from purified components or crude extracts (Cello et al., 2002). Although this method may become applicable to other viruses as the study of virus assembly leads to better in vitro assembly systems, such systems are currently not scalable for the production of larger quantities of virus, and eventually the actor would need to move into cell culture approaches. Positive strand RNA viruses, whose genomes can be directly translated by the cell to produce viral proteins, are generally easier to synthesize and boot than negative strand RNA viruses. For positive strand RNA viruses, the cDNA must be engineered to express an exact copy of the viral genome, including appropriate sequences at the 5’ and 3’ ends that govern transcription and translation, but that process is fairly straightforward. This cDNA can be transcribed in vitro to produce a viral RNA that, when transfected into cells, serves as an mRNA for production of viral replication proteins that initiate the complete viral life cycle (Kaplan et al., 1985). RNA viruses with a negative strand genome present a slightly higher challenge to synthesize because, by definition, negative strands are not translated. For these viruses, the genome is usually introduced in the cell along with an expression vector that encodes the viral replication protein(s). Then, once the cellular RNA polymerase produces the viral RNA genome from the cDNA, the viral replication machinery can take over (Neumann et al., 1999). Assuming that an actor can identify a cell line in which the virus can be grown, smaller viral genomes would be, in general, easier to boot, while large viral genomes would present a greater challenge (see Figure 4-2). Large DNA molecules must be manipulated with care to avoid fragmentation, and therefore large genomes (greater than about 30–50,000 base pairs) are subject to integrity constraints. However, overlapping DNA fragments are recombined readily once inside the cell, and in fact this ability to use the cell to stitch together fragments (Chinnadurai et al., 1979) was used extensively in the early days of gene therapy to produce adenovirus vectors expressing various transgenes. As the DNA of most DNA viruses is infectious, once that DNA enters the nucleus, the cell takes over the process of transcription and translation, ultimately leading to assembly of progeny. Poxviruses are a notable exception in that they replicate in the cytoplasm and require co-infection with a helper virus to initiate the first round of replication. The recent successful construction of the horsepox genome, which contains more than 200,000 base pairs, underscores the increasing feasibility of booting larger genomes (Kupferschmidt, 2017; Noyce et al., 2018). Usability as a Weapon (Medium-High Concern) Viruses have evolved to infect people and other organisms. The impact of a synthesized existing virus would be highly predictable based on knowledge of its natural behavior. The level of concern with regard to usability as a weapon spans a wide range depending on a particular virus’s natural tropism, virulence, environmental stability, and other such parameters. Production scale and delivery have long been considered key barriers to using existing viruses as weapons, based on knowledge of historical offensive biological weapons programs (Guillemin, 2006; Vogel, 2012). Even today, scaling up production and delivery enough to use a synthesized existing virus as a larger-scale weapon would present substantial barriers compared to a smaller- scale attack. However, the concern level is medium-high because an actor could synthesize just a small amount of virus known to be particularly dangerous, deliver it to a small number of PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 52 victims, and wait for the virus to spread as it does naturally. There are natural viruses with reproduction rates, routes of transmission, and virulence that are concerning because of the potential rapidity of spread through a targeted population after initial release or infection. Requirements of Actors (Medium Concern) The concern based on the requirements of actors is medium. The production of most DNA viruses would be achievable by an individual with relatively common cell culture and virus purification skills and access to basic laboratory equipment, making this scenario feasible with a relatively small organizational footprint (including, for example, a biosafety cabinet, a cell culture incubator, centrifuge, and commonly available small equipment). Depending upon the nature of the viral genome, obtaining an RNA virus from a cDNA construct could be more or less difficult than obtaining a DNA virus. Overall, however, the level of skill and amount of resources required to produce an RNA virus is not much higher than that for a DNA virus. There are ongoing efforts to improve the nature of the cDNA clones used to produce RNA viruses (e.g., Aubry et al., 2014; Schwarz et al., 2016), but these advances tend to be incremental in nature. The J. Craig Venter Institute (JCVI) was able to develop a viable seed stock within just three days of learning the sequence of a new strain of influenza A virus (a negative strand virus). While JCVI has extensive resources and expertise that would not be available to every actor, the demonstration nonetheless underscores current capabilities regarding booting both DNA and RNA viruses. On the other hand, one key challenge when producing some RNA viruses is the concept of quasispecies. Because viral RNA polymerases are highly error-prone, each time an RNA viral genome is copied within the cell, it generally contains one or more mutations (Lauring et al., 2012). Thus, the progeny viruses that egress from an infected cell are not a clonal population, but rather a mixture of highly related, non-identical viruses referred to as a quasispecies. The potential genetic composition of the population, therefore, is a function of the starting sequence because any given codon can only mutate to certain other codons. As most sequences deposited into databases are derived from recombinant clones, each of which represent a single member of the quasispecies, it is possible that the starting sequence may not generate a “wild type,” fully virulent population after booting. Thus, depending on the resources and expertise available to the actor, there may be difficulties in building and testing a fully virulent RNA virus. Potential for Mitigation (Medium-Low Concern) The consequence management measures for attacks using re-created known pathogenic viruses would be identical to those available for the natural pathogens, including vaccines and antivirals for some agents, along with public health measures such as social distancing and isolation of sick individuals. With current approaches, it may prove challenging to recognize and attribute such an attack because infections arising from a natural pathogen may be indistinguishable from those arising from the synthesized version. However, the same public health measures will be implemented regardless of whether the virus is synthesized or natural. While public health measures deployed to counteract natural viral outbreaks are not perfect, PREPUBLICATION COPY: UNCORRECTED PROOFS

53 Biodefense in the Age of Synthetic Biology ongoing surveillance and containment efforts in the United States are impactful and have been effective in containing some outbreaks in recent years. Screening commercially produced synthesized DNA sequences may be one of the only practical options to deter an attack using a re-created known pathogenic virus. The effectiveness of this approach, however, is undermined by the inherent limitations of list-based screening, the expectation that there are international companies that do not screen orders and are outside of U.S. regulatory control, the fact that oligonucleotides are not screened, and the fact that it is possible to synthesize genetic material in house with purchased equipment. Despite current inabilities to attribute and effectively prevent attacks using synthesized viruses, overall concern with regard to the potential for mitigation is medium-low due to the existing public health measures that could be employed against an attack. However, the concern level is higher for viruses that spread rapidly and efficiently and have a short serial interval (the time between when a person is infected with a pathogen and when he or she can spread it to others). Re-Creating Known Pathogenic Bacteria The genomes of many existing bacteria have been characterized, and the same types of DNA synthesis and booting approaches used for large viral genomes can, in theory, be applied to re- create known pathogenic bacteria. Indeed, JCVI reported the synthesis and booting of Mycoplasma mycoides in 2010 (Gibson et al., 2010). Other microbial genome synthesis projects are well underway, such as for Escherichia coli (4 million base pairs; Ostrov et al., 2016) and yeast (11 million base pairs; Mercy et al., 2017; Mitchell et al., 2017; Richardson et al., 2017; Shen et al., 2017; Wu et al., 2017; Xie et al., 2017; W. Zhang et al., 2017). The assessment of concerns related to re-creating known pathogenic bacteria is summarized here and described in detail below. Usability of Potential Usability as a Requirements the for Weapon of Actors Technology Mitigation Level of concern for Medium- Low Medium Low re-creating known low pathogenic bacteria Usability of the Technology (Low Concern) It is not yet possible to successfully re-create known bacteria; therefore, the level of concern is relatively low with regard to the usability of the technology. As is the case with viruses, GenBank® is a rich source of sequence information from which to build a known bacterium. However, given that bacterial genomes are typically 1 to 2 orders of magnitude larger than most viral genomes (see Figure 4-2), bacteria present a much greater technical challenge to synthesize and boot. In the case of the JCVI synthesis (Gibson et al., 2010), a single base pair mistake initially prevented booting of the bacteria and cost the project team months of time (JCVI, 2010). PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 54 Therefore, while the Design step is straightforward, the Build component of the Design-Build- Test cycle, in particular the construction of the full genome, currently is a significant barrier. In part, this difficulty stems from the challenge of maintaining the structural integrity of the DNA itself: DNA fragments larger than 30,000 base pairs are easily fragmented when subjected to any kind of shearing, including standard laboratory pipetting, which makes them unusable for bacterial construction. To overcome this barrier in the only synthesis of known bacteria in the literature to date, the JCVI group built the bacterial genome as a yeast artificial chromosome. FIGURE 4-2 Relative scales of genetic information encoding familiar bacteria, viruses, and toxins. NOTES: A single large toxin gene (smallest size represented in the figure, kilobase pairs) is shown in the leftmost box (lightest blue). Progressively larger genome sizes are shown in progressively darker hues moving to the right: single-stranded RNA virus genomes (kilobases), double-stranded DNA virus genomes (kilobase pairs), and bacteria (megabase pairs). The difficulty of DNA assembly and booting is partly a function of genome size and structure. SOURCE: Adapted from John Glass, JCVI. Assuming the bacterial genome can be synthesized and assembled, the next step—booting— is another particularly difficult challenge, as one cannot simply add the genome to an in vitro extract and obtain a living bacterium at the end of the reaction. Rather, the genome must be introduced into a cellular structure. The JCVI group accomplished this by transplanting their synthetic genome, propagated as a yeast artificial chromosome, into a related species of mycoplasma (Gibson et al., 2010). This transplantation approach has its own hurdles, both known (such as bacterial restriction or modification systems) and unknown. The process by which a synthetic bacterial genome may take over all necessary functions from a natural one is incompletely understood. Therefore, while obtaining the starting DNA components of a bacterial genome may be relatively straightforward from a technical point of view—they can be synthesized in house or purchased (assuming they pass or evade Select Agents screening protocols)—the subsequent assembly steps present a substantially greater challenge than with viruses. As John Glass, leader of JCVI’s Synthetic Biology and Bioenergy Group noted in a public data-gathering session during the study process, making a bacterium is “very hard and expensive.” PREPUBLICATION COPY: UNCORRECTED PROOFS

55 Biodefense in the Age of Synthetic Biology Given that the greatest bottleneck in re-creating known pathogenic bacteria is the step that moves from DNA to functioning organism, it will be important to watch for technological advances that may facilitate genome assembly and booting. For example, the development of a method to manipulate large DNA fragments without physically damaging them could reduce the difficulty of assembly. Or if a technique were developed that allowed direct transfer of the bacterial chromosome from the yeast in which it was built into a bacterial host, this would overcome the hurdles of shearing and transplantation. However, yeasts are not known to even transfer chromosomes among themselves, except during mating; therefore, such a yeast-bacterial system would likely need to be developed from scratch if this approach was going to be pursued. Usability as a Weapon (Medium Concern) If a pathogenic bacterium were successfully synthesized, its properties as an infectious agent would be predictable based on the known properties of the naturally occurring bacterium. As with synthesized viruses, the level of concern therefore depends on the bacterium’s natural tropism, virulence, environmental stability, and other such parameters. As with viruses, scaling up production and delivery enough to use synthesized bacteria as a weapon of mass destruction would present substantial barriers compared to a smaller scale attack, raising many classical weaponization issues such as environmental stability during mass dispersal. Overall, the level of concern related to usability as a weapon is medium, but there is a wide range of concern with regard to different bacterial pathogens, reflecting differences in the potential for weaponization of various types of bacteria in general. For example, a bacterium that forms spores should be easier to disperse throughout, and would be more stable in, the environment compared to a bacterium that does not form spores. Requirements of Actors (Low Concern) Making an existing bacterium from scratch currently is very difficult and requires substantial expertise and resources—significantly more resources than would be required to synthesize a known virus. Therefore, concern on this factor is relatively low. An actor would need specialized, hands-on experience working with large bacterial genomes, a level of sophistication that takes years to achieve and is currently rare. In addition, this work would require a large amount of money and a fairly long time, as evidenced by the experience of groups working in this area, such as JCVI.6 This would likely necessitate a large organizational footprint. Thus, the capability to both construct and boot such genomes is likely to remain accessible only to large, multidisciplinary teams that have access to substantial resources (funding, equipment, diverse and well-developed skill sets) for at least the next five years. 6 The 2010 creation of the synthetic Mycoplasma mycoides bacterial cell by JCVI reportedly took 15 years and cost $40 million to accomplish (see Sleator, 2010, and JCVI, 2010). PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 56 Potential for Mitigation (Medium-Low Concern) Overall, concern with regard to the potential for mitigation is medium-low due to the well- established response options that are in hand for known bacteria. In terms of consequence management, there is a wide array of antibiotic drugs that could be used to contain attacks using bacterial pathogens (indeed, a wider array than the number of antivirals available). However, antibacterial drug resistance can be expected to limit the number of drugs that would be effective in any given case, and the re-creation of a highly virulent, antibiotic-resistant bacterium capable of aerosol transmission would pose greater concern. In terms of prevention, it would be extremely difficult, if not impossible, to distinguish a facility being used to develop bioweapons based on synthesized pathogenic bacteria from a legitimate academic or commercial facility. The Federal Select Agent Program may provide some deterrence for these activities within the United States, although screening protocols leave many loopholes that could allow for the undetected synthesis of bacterial genome fragments for Select Agents. Also, considerations related to recognizing and attributing an attack using synthesized bacteria are identical to those for synthesized viruses; it may be quite difficult to distinguish infection by a natural pathogen from that arising from the synthesized version. MAKING EXISTING PATHOGENS MORE DANGEROUS The age of synthetic biology has enabled the manipulation of viruses and bacteria to alter their genotypes, and therefore, their phenotypes. The gene therapy field has made engineering the tropism of viruses an active area of research, and bacteria are commonly manipulated to serve as a platform for the production of useful compounds. These same experimental approaches could be used to develop new weapons. Traits of viruses and bacteria (both pathogenic and nonpathogenic) that could potentially be modified to engineer bioweapons— along with current technological capabilities and anticipated future developments relevant to pursuing such activities—were considered in assessing the level of concern warranted for the potential use of synthetic biology to make existing pathogens more dangerous. Making Existing Viruses More Dangerous An actor seeking to make an existing nonpathogenic virus pathogenic or an existing pathogenic virus more dangerous or better suited for a biological attack would have multiple routes to consider. There are already some examples in the literature in which the use of biotechnology has resulted in a virus with enhanced virulence, an expanded host range, or other features that make it more pathogenic. In analyzing the level of concern warranted for this type of activity, a number of viral traits that potentially could be attempted using synthetic biology or standard techniques were considered (see Box 4-1). PREPUBLICATION COPY: UNCORRECTED PROOFS

57 Biodefense in the Age of Synthetic Biology BOX 4-1 Viral Traits The following are selected examples of viral traits, presented to give a sense of the range and type of traits that could theoretically be targeted for modification using biotechnology. Altered Tropism Tropism is the capacity of a virus to infect or damage specific cells, tissues, or species. While tropism is primarily influenced by the interaction of the viral cell attachment protein(s) with the receptor(s) present on the cell (thus determining viral entry), the larger property of tropism is determined by multiple viral and host cell factors (Heise and Virgin, 2013). Altering tropism could be used to expand the host range of an existing virus or otherwise increase a virus’s ability to take hold in a targeted population. Several studies have demonstrated the ability to alter the tropism of viruses. The avian influenza H7N9 strain has been causing isolated human infections since the initial outbreak in China in 2013, but sustained human-to-human transition has not been documented. In a recent publication, de Vries and colleagues (2017) demonstrated that only three mutational changes in the sequence of the hemagglutinin gene are sufficient to switch the virus’s tropism from avian to human and support binding to human tracheal epithelial cells. However, the researchers did not perform follow-up experiments to test whether these mutations were sufficient to make an actual host range shift in the ferret model. In earlier studies with avian influenza, researchers used site-directed mutagenesis to introduce mutations into the hemagglutinin gene to allow wild-type H5N1 virus to bind to human receptors (Herfst et al., 2012). This group went on to show that as few as five mutations can lead to airborne transmissibility of H5N1 between ferrets (Linster et al., 2014). Researchers have also used synthetic biology to alter tropism in investigations of the respiratory syndromes SARS and MERS. There is considerable evidence indicating that a SARS-like virus in bats was the origin of the 2003 outbreak of SARS in humans (Li et al., 2005). The bat virus, however, does not grow in cell culture. To help elucidate the steps that may have occurred to convert bat SARS-CoV into a virus infecting humans, Becker and colleagues (2008) substituted the human SARS coronavirus receptor binding domain for the equivalent domain in the bat SARS-CoV virus, making the bat-SARS virus replication competent in cell culture and mice. Similarly, to develop a small-animal model of MERS- CoV, researchers modified both the mouse, to express a chimeric receptor, and the virus (Cockrell et al., 2016). Enhanced Viral Replication Enhancing viral replication could help increase the impact and spread of a virus-based bioweapon. In experiments with echovirus 7, Atkinson and colleagues (2014) demonstrated that decreasing the CpG and UpA frequencies in two 1.1 to 1.3 kilobase regions of the viral genome enhanced viral replication in susceptible cells. Conversely, increasing the CpG and PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 58 UpA frequencies resulted in decreased viral replication. While it is unknown whether these results would be the same in animals—enhanced replication in cell culture does not necessarily correlate with enhanced replication in vivo, and in fact, the reverse is sometimes the case—an actor with sufficient time and resources may be able to generate variants empirically and passage them in a susceptible host to select a variant with enhanced replication ability. Enhanced Virulence Virulence measures the relative capacity of a virus to cause actual disease in a host, rather than just infection. Virulence represents the combined effect of multiple genes and determinants that play specific roles in specific settings in vivo (Heise and Virgin, 2013). In the best known example of an engineered virus resulting in enhanced virulence, Jackson and colleagues (2001) engineered ectromelia virus (mousepox), a member of the orthopoxvirus genus and a natural pathogen of mice, to express mouse interleukin-4 (IL-4), with the goal of producing a contraceptive vaccine to control the mouse overpopulation. In the mouse model the recombinant virus was shown to suppress primary antiviral cell-mediated immune responses and overcome pre-existing immunity. It is also conceivable that actors would seek to manipulate a virus so that it causes disease by different mechanisms than a natural virus might, such as by manipulating neurobiology or altering the host microbiome. Ability to Evade Immunity At the root of the increased virulence demonstrated in the mousepox experiments (described under Enhanced Virulence, above) was the recombinant virus’s capability to evade immunity. This points to another potential route for actors seeking to produce bioweapons: the development of viruses designed to anticipate and evade the immune response or even to overcome vaccine-based immunity. Detection of viral pathogens by the innate immune system leads to the induction of antiviral mechanisms that are mostly mediated by type-1 interferons. This primary response then leads to the activation of the adaptive immune response that is more directed, antigen-specific, and long-lasting (Iwasaki and Medzhitov, 2013). Many viruses have countermeasures to subvert the innate immune response including interferon- induced antiviral activity (Chan and Gack, 2016, for review). It may be possible to express one or more antagonists of these antiviral activities in a pathogen that does not already have that particular antagonist. In this way, the arsenal of activities that a virus uses to evade the innate immune response would be expanded and virulence may be enhanced. The creation of chimeric viruses developed by genetically substituting capsid genes has been well documented (Guenther et al., 2014, for review). These viruses have mainly been developed in the context of, for example, improving adenovirus vectors to target specific tissues and as an approach to circumventing pre-existing viral immunity that may limit the use of viral gene therapy vectors (Roberts et al., 2006). It is conceivable that the latter approach could be used to develop a chimeric viral vector expressing a toxin gene targeted to a particular tissue and used in a population with pre-existing immunity to the vector virus. The PREPUBLICATION COPY: UNCORRECTED PROOFS

59 Biodefense in the Age of Synthetic Biology molecular determinants of targeting are poorly understood, however, and these approaches generally require significant trial and error to be successful. Ability to Evade Detection Some modifications could result in a virus that would be difficult to detect using current outbreak response approaches. The most commonly used methods of laboratory identification of viruses are based on real-time Polymerase Chain Reaction (PCR) assays in which specific primers and fluorescently labeled probes are designed to bind to conserved and unique regions of the viral DNA or cDNA. Non-targeted methods of detection include array-based assays and next-generation sequencing, but these are not yet in wide use in clinical and commercial laboratories. Cell culture methods are rapidly disappearing from use. Mutations that target the primer binding sites could therefore result in a virus that is not recognizable. Ability to Resist Therapeutics Actors could seek to develop viruses capable of resisting available therapeutics, though the necessity of this approach would depend on whether effective therapeutics exist. Despite the availability of successful antiviral agents such as those used to counter HIV (human immunodeficiency virus), herpes viruses, influenza viruses, and HCV (hepatitis C virus), there are no specific antiviral drugs for the vast majority of viruses. Even where antivirals exist, the development of resistance to these drugs is almost inevitable unless the rate of replication of the virus in the presence of the drug can be completely inhibited or, alternatively, if multiple drugs are used in combination against different viral targets (Coen and Richman, 2013). For example, newer antivirals based on immune inhibition, such as the ZMapp therapeutic, are a mixture of three humanized monoclonal antibodies developed against Ebola virus and have shown survival benefits in non-human primates experimentally infected with the virus (Pettitt et al., 2013). A randomized, controlled trial in humans appeared to show beneficial effects but did not meet the prespecified statistical threshold for efficacy (Davey et al., 2016). Enhanced Transmissibility Airborne transmission of pathogens occurs through aerosolization and droplets. Airborne transmissibility determines the distance over which the virus may travel, and the determinants of this property are complex and dependent on multiple host and viral factors (Herfst et al., 2017). In a follow-up to the H5N1 experiments described under Altered Tropism (above), the mutated virus was sequentially passaged in ferrets to force natural selection of heterogeneous viral mixtures and, after 10 passages, naïve recipient ferrets were exposed to the infected ferrets in an adjacent cage without direct contact. Three of four recipient ferrets became infected, demonstrating that selection had occurred for airborne transmissibility of the virus (Herfst et al., 2017). In another study, Imai and colleagues (2012) constructed a reassortant PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 60 virus possessing the hemagglutinin from an H5N1 virus and seven gene segments from a 2009 H1N1 virus. After passaging through ferrets, a mutant of this reassortant was obtained that had four mutations in the hemagglutinin gene and was capable of respiratory droplet transmission in ferrets. This work demonstrated that a mammalian transmission phenotype could be conferred to highly pathogenic H5N1 influenza. Enhanced Stability The stability of a virus outside the host is influenced by multiple environmental factors including temperature, ultraviolet radiation, relative humidity, and air movement, as well as the structure of the pathogen itself. Enveloped viruses are generally less stable outside the host than non-enveloped viruses (Polozov et al., 2008; Herfst et al., 2017). While it would be impossible to convert an enveloped virus to a non-enveloped virus because addition of the envelope is tightly coupled to specific features of the replication cycle, it may be possible to alter other features of a virus to enhance its stability for weaponization and mass dispersal. Reactivation of “Dormant” Virus It may be possible to use chemical or biological means to reactivate latent or persistent viruses. Such an attack could be targeted based on whatever endogenous mix of pathogens already exists in an individual or population. For example, some viruses, like HCV, cause chronic infections whose clinical symptoms do not appear until late in life; developing a chemical or biological trigger to accelerate the pathogenesis of such a virus is a possibility. It may even be possible to recombine a modern virus that has little pathogenicity and spreads widely with an earlier, perhaps more deadly, endogenous variant. Lower immunity in hematopoietic stem cell transplant patients has been shown to result in widespread viral reactivation, sometimes life-threatening (Cavallo et al., 2013), underscoring the potential impact of such approaches. Research focused on coaxing HIV out of latent reservoirs in order to completely cure the infection, the so-called “shock and kill” strategy (Shirakawa et al., 2013), could further advance potential dual use research in this area. The assessment of concerns related to making existing viruses more dangerous is summarized here and described in detail below. Usability of Usability as a Requiremen Potential for the Weapon ts of Actors Mitigation Technology Level of concern for making Medium- existing viruses more Medium-low Medium Medium high dangerous PREPUBLICATION COPY: UNCORRECTED PROOFS

61 Biodefense in the Age of Synthetic Biology Usability of the Technology (Medium-Low Concern) Overall, the usability of the technology required for this capability involves many barriers, leading to an assessment of medium-low concern for this factor. Although scientists have a strong understanding of viruses and their biology and can conceive of many ways to manipulate them, modifying viral characteristics intentionally using rational design remains a substantial challenge. In most cases, the viral phenotype is a result of many interrelated viral functions resulting from a diverse array of genetic networks as well as host and environmental factors. Good examples of this complex situation are found in the reviews of Herfst et al. (2017) and Plowright et al. (2017), which discuss drivers of airborne transmission and zoonotic spillover, respectively. Rarely can a specific phenotype be attributed to a single gene, or an altered phenotype to a specific mutation. Furthermore, the determinants of tropism, transmissibility, and other properties are often not well understood or predictable. Many of the research advances achieved to date have involved significant trial and error (e.g., gene therapy vector tropism modifications [Nicklin and Baker, 2002]), inadvertent findings (e.g., the outcomes of IL-4 expression in ectromelia virus [Jackson et al., 2001]), or directed evolution (e.g., experiments altering transmissibility of avian influenza virus (Herfst, 2012; Imai et al., 2012). How these alterations would affect the behavior of these viruses in the human population is difficult to assess because of limited knowledge regarding how genotype would translate to phenotype, but a successful introduction of such a modified virus into humans could have dire consequences. Although this knowledge gap of how to engineer complex viral traits is likely to limit the ability to engineer viruses for enhanced bioweapons currently, it will be important to monitor for developments that significantly increase the ability to relate genotype to phenotype—the knowledge of determinants of complex viral traits and how to engineer pathways to produce them. An added barrier is that introducing mutations into a viral genome almost invariably results in an attenuated (i.e., less pathogenic) virus (Holmes, 2003; Lauring et al., 2012), as there are constraints on viral genome organization. The introduction of mutations has been the classical method of making many effective live attenuated vaccines, including those for measles and yellow fever, as well as the Sabin poliovirus vaccine strain (Sabin, 1985). The mutation(s) in these examples were introduced in a non-directed manner by passage in cell culture and resulted in phenotypic changes that lessened the virus’s ability to cause a harmful infection. An exception to this assessment of medium-low concern, however, would be the introduction of antiviral resistance. It is more feasible to introduce mutations that allow resistance to antivirals without causing attenuation, because the exact point mutations responsible for drug resistance are often known and generally do not lead to significant attenuation. The majority of alterations in a viral genome can be performed with standard recombinant DNA technology methods and do not require advanced synthetic biology techniques. One exception is the multiple substitutions required to change the frequency of particular bases to make synonymous mutations at multiple positions. Achieving this would be much simpler with the large pieces of DNA that synthetic biology technologies assist in producing, as well as synthetic biology tools that allow for the introduction of mutations in a directed manner and the application of many mutations simultaneously. For example, researchers are now using synthetic biology to introduce many synonymous mutations (including alterations in a DNA or RNA sequence that do not change the protein amino acid sequence), in an effort to make live attenuated viral vaccines that have better genomic stability (Wimmer et al., 2009, Martinez et al., PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 62 2016). Given the precision required and the limitations of rational design, an alternative approach would be to use combinatorial libraries, high throughput screening, or directed evolution to test many candidate modifications. For example, viruses could potentially be tailored to evade specific immune responses by using computational modeling, high-throughput screening, or directed evolution to escape the most likely or most capable antibodies or T-cell receptors, provided that immune-dominant epitopes on a pathogen are known. However, even this approach would be constrained to some extent by the amount of available information regarding the determinants of the target phenotype and potentially by the current size limits of combinatorial libraries. It is not possible to test an infinite number of variations, although with available technologies a well-resourced actor would be capable of testing quite a lot. Finally, in addition to developing the variants to test, it is necessary to boot the recombinant genome in a cell line. Depending on the virus, this booting step can present a significant barrier, and booting imposes additional limits on the number of variants that can feasibly be tested. Usability as a Weapon (Medium-High Concern) Because viruses have certain characteristics consistent with use as a weapon, and because the modification of the virus may enhance those characteristics, the concern is medium-high for this factor. Just as the types of manipulations required to alter the phenotype of a virus are difficult to predict, how a modified virus will behave when introduced into the human host is also difficult to anticipate. In addition, the tendency for alterations to attenuate viruses may serve as a “natural” mitigating factor and reduce the effectiveness of a bioweapon produced in this way. Testing modified viruses may also present a barrier (unless the actor is willing to test in humans). For example, animal models do not always predict how a virus will behave in humans. It has been argued that avian influenza virus transmission in ferrets does not mean with certainty that those viruses would also transmit from human to human via an airborne route (Racaniello, 2012; Lipsitch, 2014; Wain-Hobson, 2014), but as noted above, if an engineered virus does acquire this property, the dynamics of weapons use change. If modifications are pursued with the intention of making the virus more dangerous in some way, the scope of casualty for an attack using a modified virus could be larger than an attack using a natural virus. If the modifications are intended to make the virus easier to produce or deliver, the resulting virus may bypass some of the classical barriers to weaponization, such as environmental stability during mass dispersal. Otherwise, a modified virus would present many of the same weaponization opportunities and challenges as those detailed for the re-creation of a known pathogenic virus. Requirements of Actors (Medium Concern) Modifying a virus would require excellent molecular biology skills and advanced knowledge of the field. Understanding and being able to verify the product therefore imposes an expertise barrier to successfully manipulating viral phenotypes. In general, however, the resources and organizational footprint required would be moderate, similar to those required for re-creating a known pathogenic virus. Therefore, there is a medium level of concern with regard to this factor. PREPUBLICATION COPY: UNCORRECTED PROOFS

63 Biodefense in the Age of Synthetic Biology Potential for Mitigation (Medium Concern) Existing tools for mitigation, such as public health systems and antivirals, may be effective against a modified virus. However, in general, they would be expected to be less effective against modified viruses than against the naturally occurring ones for which they are designed, leading to a medium level of concern for this factor. In particular, available medical countermeasures may be ill-suited against viruses with modifications designed to confer antiviral resistance or to alter the ability of the virus to be recognized by the immune system. Diagnostic approaches using sequencing would be effective for identifying a modified virus as being laboratory-derived in the vast majority of cases (antiviral resistance being one notable exception), but it is unclear whether that capability would effectively facilitate attribution. While the overall level of concern for this capability is medium with regard to the potential for mitigation, the concern level is higher for viruses with pandemic potential, such as influenza, for which a modified virus could present significant challenges in terms of measures to limit spread or reduce impact. Making Existing Bacteria More Dangerous As with viruses, an actor seeking to make an existing nonpathogenic bacterium pathogenic or to make an existing bacterial pathogen more dangerous would have many potential routes to consider. In analyzing the level of concern warranted for this type of activity, a number of modifications to existing pathogenic or nonpathogenic bacteria that potentially could be attempted using biotechnology were considered. Box 4-2 notes some of the ways in which such activities might differ in the context of bacteria compared to viruses. BOX 4-2 Bacterial Traits The following are selected examples of bacterial traits, presented to give a sense of the range and type of traits that could theoretically be targeted for modification using biotechnology. This box focuses on how modifying traits in bacteria might differ from modifying analogous traits in viruses, described in Box 4-1. Altered Tropism Unlike viruses, which are exclusively intracellular pathogens, bacterial pathogens can be either intracellular or extracellular. Generally, extracellular pathogens are relatively environmentally stable and good at adapting to their environment. Even those that are not spore-forming often have the capacity to replicate and cause damage in multiple tissues and cell types and in different locations in the body. Given their environmental stability, they are difficult to eradicate and may not require host-to-host contact for transmission. Intracellular bacteria, like viruses, rely on host cell nutrients and are often able to evade the host immune system (Finlay PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 64 and McFadden, 2006). Intracellular pathogens are usually transmitted via direct contact or aerosol transmission. Both intracellular and extracellular pathogens rely on adherins and colonizing factors, which facilitate contact with host target cells, confer resistance to leukocyte attack, and are significant virulence factors (Ribet and Cossart, 2015). Enhanced Virulence Many factors influence bacterial virulence and could potentially be targeted for modification. The primary mechanisms of bacterial pathogenesis include host target cell death (Böhme and Rudel, 2009), whether by cell lysis (resulting either from the multiplication of intracellular pathogens or as a result of the action of bacterial toxins) or by induction of apoptosis (programmed cell death); mechanical perturbations of host physiology (e.g., blockage of circulatory or respiratory passages due to the size or number of invading bacterium or as a result of mucous production); host cell damage resulting from the host immune response to the bacterial infection; and the action of bacterial toxins. The effects of cell death depend upon the host cells involved and are influenced by the bacterial burden introduced, the route of infection, complicating symptoms induced by host immune response, and the rapidity of the infection process. Colonization potential is influenced by the ability of some pathogenic bacteria (e.g., Shigella) to trigger premature or unscheduled apoptosis in the host cells they infect (Gao and Kwaik, 2000); the initial phase of this process involves the introduction of enzymatically driven damage to host cell DNA followed by massive disturbances in cell integrity and cell death. Another significant virulence factor is the ability of some bacteria (e.g., Bacillus anthracis) to form capsules consisting of polysaccharides and amino acids (Cress et al., 2014). Capsules prevent bacteria from being phagocytized by neutrophils and macrophages. Other virulence factors include invasion factors, which are usually encoded chromosomally but may also be plasmid-borne, and siderophores, iron-binding factors that allow bacteria to compete with host cells for iron acquisition (Quenee et al., 2012). Enhanced Toxin Production Many bacterial pathogens cause damage to host cells and tissues through the production of toxins. These toxins take two forms: exotoxins and endotoxins. Exotoxins are relatively unstable, highly antigenic proteins that are secreted into host body fluids. Some exotoxins are bound to the bacterial cell wall following their synthesis and are released upon lysis of the invading bacterium (Sastalla et al., 2016). Often highly toxic, exotoxins are produced by both Gram positive and Gram negative bacteria. Some exotoxins can act only on certain cell types while others affect a broad spectrum of cells and tissues. Some bacterial pathogens make only a single toxin (e.g., cholera, diphtheria, tetanus, botulism) while others can synthesize two or more distinct toxins (e.g., staphylococcus, streptococcus). Antitoxin antibodies to exotoxins are usually made rapidly by the host. The genetic determinants of exotoxins are often found on extrachromosomal elements, usually plasmids or bacteriophages. Endotoxins, on the other hand, are relatively stable, lipopolysaccharide components of the outer membrane of some Gram negative bacteria that can act as toxins under certain PREPUBLICATION COPY: UNCORRECTED PROOFS

65 Biodefense in the Age of Synthetic Biology circumstances (Zivot and Hoffman, 1995). The Lipid A appears to be the toxic component, which can act while in the intact bacteria expressing it. Endotoxins are generally weakly immunogenic, eliciting fever in the host. They can cause hypotension due to increased vascular permeability accompanied by vasodilation, which can in turn result in shock. The genetic determinants for endotoxins are chromosomal. Actors could potentially seek to modify bacteria to enhance their natural toxin production or introduce toxin production into a bacterium that does not naturally produce toxins. Such approaches are further discussed in chapter 5. Ability to Evade Immunity As with viruses, it is possible to engineer bacteria to anticipate or evade the immune response. Ability to Evade Detection As with viruses, the most commonly used methods of laboratory identification of bacteria are based on real-time PCR assays in which specific primers and fluorescently labeled probes are designed to bind to conserved and unique regions of the bacterial chromosomal or extrachromosomal DNA. Another widely used method in clinical microbiology laboratories is MALDI-ToF (matrix-assisted laser desorption/ionization time-of-flight), a method of ionizing large molecules and identifying them by mass spectrometry in comparison to reference standards. Non-targeted methods of detection such as array-based assays and next-generation sequencing are available but are not yet in wide use in clinical and commercial laboratories. Culture methods are rapidly disappearing from use (Carleton and Gerner-Smidt, 2016). Ability to Resist Therapeutics In contrast to the relatively small number of antivirals, there are many antibacterial agents available that are capable of acting against a wide variety of bacterial pathogens. However, bacteria can be intrinsically resistant to antibiotics, or can acquire resistance via chromosomal mutation and horizontal gene transfer. There are three main mechanisms of antibiotic resistance (Blair et al., 2015). First, the bacterium can prevent the antibiotic from accessing its target, either through reduced permeability of the antibiotic through the cell wall or membrane complex, or through increased efflux of the antibiotic back out of the organism and away from its target. Second, the antibiotic target can be altered through genetic mutation, causing the target to become modified or protected. Finally, antibiotic resistance can be acquired by direct modification of the antibiotic itself, either by inactivation by antibiotic hydrolysis or by way of inactivation due to a chemical modification. These mechanisms are well studied and could potentially be adapted for the purposeful creation of antibiotic-resistant pathogenic bacteria. PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 66 Enhanced Transmissibility As with viruses, the property of airborne transmission in bacteria is complex and dependent on multiple host and pathogen factors, in particular environmental stability and tissue tropism. Extracellular bacterial pathogens are extremely adaptable to environmental challenges and may not require host-to-host contact for transmission, making these pathogens difficult to eradicate. In addition, many bacterial pathogens that replicate extracellularly are capable of causing damage to different cells and tissue types. On the other hand, many intracellular bacterial pathogens are communicable (i.e., capable of host-to-host transmission), facilitating rapid spread within a community and thus presenting a greater capacity to threaten public health. Enhanced Stability The environmental stability of a bacterium depends on its physiology and life cycle. Generally, due to the composition and structure of cell walls, Gram positive bacteria are more environmentally stable than Gram negative bacteria. In addition, when subjected to harsh environmental conditions such as desiccation, some Gram positive bacteria form spores capable of remaining viable in the environment for decades, albeit in a metabolically dormant state. For example, spores of Bacillus anthracis can remain viable in the environment for up to a century (Friedmann, 1994; Repin et al., 2007; Revich and Podolnaya, 2011) and constitute the infectious form of this pathogen (with vegetative forms not being infectious). Actors may find it advantageous to engineer bacterial cell walls to more closely resemble Gram positive organisms to enhance survival during aerosol dissemination and allow the agent to remain viable and available to infect the target host for extended periods of time. The assessment of concerns related to making existing bacteria more dangerous is summarized here and described in detail below. Usability of Potential Usability as a Requirements the for Weapon of Actors Technology Mitigation Level of concern for High Medium Medium Medium making existing bacteria more dangerous PREPUBLICATION COPY: UNCORRECTED PROOFS

67 Biodefense in the Age of Synthetic Biology Usability of the Technology (High Concern) Generally speaking, the technology requirements for making existing bacteria more dangerous are relatively low, which leads to a relatively high level of concern for this factor. While it is technically difficult to design and build bacteria from scratch, altering existing bacteria is relatively easy with molecular and genetic approaches. These capabilities make the Design phase of the Design-Build-Test cycle relatively straightforward, especially if the desired trait is conferred through a well-elucidated gene or pathway, such as known genes for antibiotic resistance or toxin production. In terms of the Build step, there are well-established techniques to insert, delete, or change existing genes (Selle and Barrangou, 2015; Wang et al., 2016; H. Zhang et al., 2017). Making such modifications does not necessarily require synthetic biology approaches, though such technologies can enhance the process. Some bacterial species are easier to manipulate genetically than others. In general, this step is easier if the genetic changes are smaller in size or fewer in number and more difficult for larger or more extensive modifications. In addition, if a desired pathogen has a close nonpathogenic relative, a researcher could splice relevant portions of the pathogen’s genome into the genome of the relative. In general, it is easier to manipulate bacteria than viruses. In part, this is due to the relative sizes of bacterial versus viral genomes; for viruses there are fitness pressures and constraints on genome packaging to keep the genome smaller, thus tending to attenuate modifications over time. Modifications are more likely to persist in a bacterial genome because those genomes are genetically more stable. In viruses, enhancement of one phenotype often results in diminution of another, a factor that would likely be difficult to overcome in viruses but presents less of a barrier when modifying bacteria. Some types of bacterial modifications would be easier to achieve than others; engineering bacterial traits that are complex requires greater knowledge of trait determinants and how to engineer pathways to produce them. On the more difficult end of the spectrum is altering tropism, which involves the complex interplay of a multitude of bacterial genes that are fundamental to the physiology of a specific bacterium (Pan et al., 2014). Tropism in bacteria is less likely to be alterable using synthetic biology approaches compared to tropism in viruses; however, there are routes that could be pursued. Both intracellular and extracellular bacterial pathogens rely on adherins and colonizing factors to facilitate contact with host target cells (Ribet and Cossart, 2015). It may be feasible to use synthetic biology technologies and big data analytical capabilities to engineer and express novel adherin or colonizing factor analogues of these bacterial proteins and introduce them either by encoding them on episomes or integrating them into the chromosome. Given the complexity of the host-pathogen interaction, transmissibility and communicability of bacterial pathogens in humans would also be difficult to confer or alter. In a similar vein, it would be challenging to manipulate a bacterial pathogen to acquire efficient airborne transmission. Among other characteristics, the pathogen’s success would depend on environmental stability, which is intrinsic to its physiology and life cycle. It is not yet technically possible to alter a bacterial pathogen’s environmental stability in a fundamental way, such as by converting a Gram negative bacterium to Gram positive or a non- spore-forming bacterium to a spore-forming bacterium. That said, synthetic biology approaches would have greater likelihood for success in this realm than would standard molecular biology approaches. On the other hand, bacterial toxins, both endotoxins and exotoxins, are clearly significant virulence factors that can likely be readily modified or designed based upon data analysis. Given PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 68 that endotoxins are chromosomally expressed and are intrinsic to the physiology of the bacterium in question, an actor would likely need to use a combination of synthetic biology and standard molecular biology approaches to modify existing endotoxins or create new ones. In addition, it is relatively trivial to confer resistance to antimicrobial drugs via standard molecular biology technologies (as demonstrated by the fact that it was done many years ago [Steinmetz and Richter, 1994]), and synthetic biology approaches would further enable targeted mutations to create a drug resistance phenotype. Usability as a Weapon (Medium Concern) The weaponization potential for making a bacterial pathogen more dangerous is, overall, of medium concern. Historically, scale-up and environmental stability have been key barriers to the weaponization of bacteria. Synthetic biology does not drastically change this equation. Despite a sophisticated understanding of some traits, such as antibiotic resistance and toxin production, knowledge is still limited for traits relevant to production and delivery of bacteria as a bioweapon, as noted under Usability of the Technology, above. Requirements of Actors (Medium Concern) The expertise required to design genetic modifications to affect bacterial traits varies widely depending on the nature of the modification (for example, those that change the bacterium’s biology in a new way would be more challenging) and the amount of available information about the genes involved (for example, those involved in toxin production and antibiotic resistance are fairly well elucidated and would thus be accessible to someone with less expertise). Thus, as more information is published relevant to more traits, the level of expertise required to design modifications to those traits is reduced. Based on the current state of knowledge, this factor poses a medium level of concern. Making the actual modifications would require classical molecular biology expertise and experience in bacterial genetic approaches, but does not necessarily require training in advanced synthetic biology techniques. Potential for Mitigation (Medium Concern) The current concern level for this factor is medium. As discussed in the context of re-creating known pathogens, the Select Agents list and voluntary screening guidelines are not likely to be sufficient to deter or prevent the development of modified bacterial pathogens. In terms of consequence management, one fundamental difference between responding to a naturally occurring new organism that has unique characteristics and responding to a modified bacterial pathogen that is a purposefully deployed biological weapon is a calculating adversary. While public health system components such as the National Syndromic Surveillance Program (NSSP) of the U.S. Centers for Disease Control and Prevention may indeed be well suited to detecting PREPUBLICATION COPY: UNCORRECTED PROOFS

69 Biodefense in the Age of Synthetic Biology and containing new naturally occurring bacterial threats, an engineered organism resistant to antibiotics will challenge the ability of public health systems to contain and respond to such a pathogen. Thus, consequence management capabilities would be less effective in the face of bacterial pathogens engineered specifically to evade them, such as through resistance to vaccines or antibiotics. CREATING NEW PATHOGENS A major aspiration within the field of synthetic biology is the design and creation of new organisms with beneficial uses. In the context of bioweapons, the possibility that this aspiration may potentially be directed toward producing pathogens that are entirely new was considered. In contrast with the discussion of modifying existing pathogens, the term “new” is used here to describe novel combinations of genetic parts from multiple organisms for which the product is not recognizable as primarily from one source. This can include genetic parts designed computationally with no near relative in the natural world. The resulting range of potential bioweapons in this category is extremely broad, but serves to illustrate the more challenging applications that may be possible at some point in the future. One example of a new pathogen would be a virus constructed from parts of many different natural viruses. This mix-and-match approach might be used to combine the replication properties of one virus, the stability of another, and the host-tissue tropism of a third, for example. A variety of experimental approaches would be applicable to this goal. Directed evolution approaches could be used to sample random combinations of viral DNA parts; while each individual combination would have a small chance of success, sampling a very large number of combinations would increase the chances of success. More explicit design approaches might be to develop software to model and predict the properties of specific designs, which would then be built, tested, and improved through multiple iterations of the Design-Build-Test cycle. As discussed under Making Existing Viruses More Dangerous, however, even simple changes to existing viruses can produce drastic deficiencies in key viral properties, making any such effort especially difficult. Nonetheless, work involving re-composing the structure of a bacteriophage genome into modular pieces (Chan et al., 2005) suggests that radical new combinations of viral sequences may be viable, though tools to design viruses with high confidence of success are currently lacking. A different example of a new pathogen would be one based on synthetic “genetic circuits” (described in Appendix A). A major pursuit within synthetic biology is the capability to arbitrarily program specific functions using genetic material. These efforts are exemplified by the engineering of DNA-encoded programs, relying heavily on concepts derived from information theory and computer science, such as constructing logic gates from individual switching functions. Importantly, the genetic material encoding those functions can in principle come from anywhere—from any branch of the tree of life or from an entirely new DNA sequence that has never been observed in nature. The designs for genetic circuits have greatly increased in complexity over time (see Toman et al., 1985, for an early example) through increased reliance on component abstractions and standardization. Figure 4-3 shows a recent example of software developed to enable such advanced designs in general, but not specifically in the context of pathogens. PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 70 FIGURE 4-3 Illustration of genetic circuit engineering facilitated by a software environment that couples circuit specification and design to predictive models of circuit function. NOTE: Genetic circuits are a common staple for work in synthetic biology and allow users to combine multiple functions from the broad categories of sense, compute, and actuate. SOURCE: Nielsen et al., 2016. While a number of genetic circuits have been designed to function in human cell lines in culture, applications using genetic circuits in the human body are still in their infancy (Lim and June, 2017). The potential for using such technology to cause harm in the human body is thus a subject of broad speculation. Novel circuits could (in theory) be used to convert a healthy cell into a cancerous one or to provoke an autoimmune response. Such circuits might be designed to act on the host DNA using engineered factors that turn host genes on or off, such as at the level of transcription or translation. A variety of mechanisms have been demonstrated for such general-purpose switching, including the use of natural or artificial microRNA molecules and the use of CRISPR/dCas9-type programmable gene repression or activation (Luo et al., 2015). Importantly, these are examples of mechanisms that have displayed a high degree of programmability in terms of which host DNA sequences can be targeted. In a similar vein, the potential programmability of genetic effectors may also lead to genetic circuits that sense and compute based on the state or type of cell (Weiss et al., 2003) or even specific genetic identity. In some cases, genetic circuits could be delivered to a small number of host cells using non- replicating delivery mechanisms, which could be either virus-derived, such as those used in some gene therapies (see chapter 7, Gene Therapy), or based on non-biological materials. At the extreme end of difficulty (and feasibility) lies the engineering of life forms that are particularly dissimilar from known life on this planet. “Xenobiology” (described in Appendix A) offers some possibilities—for example, a bacterium employing a different combination of deoxyribonucleotides and ribonucleotides to encode its genetic information (Y. Zhang et al., 2017). There is a wide range of expert opinion as to the long-term plausibility of such efforts. The assessment of concerns related to creating new pathogens is summarized here and described in detail below. PREPUBLICATION COPY: UNCORRECTED PROOFS

71 Biodefense in the Age of Synthetic Biology Usability of Usability as a Requiremen Potential for the Weapon ts of Actors Mitigation Technology Medium- Medium- Level of concern for Low Low high high creating new pathogens Usability of the Technology (Low Concern) Because the creation of new pathogens faces multiple major knowledge and technical barriers, including knowledge regarding minimal requirements for virus and bacteria viability and the constraints on viral organization discussed above, the level of concern for this factor is very low at present. However, this is a clear example of an area that warrants ongoing attention. If the technical barriers can be overcome in the future, the level of concern would increase substantially. For example, the recent engineering of a designed nucleocapsid (a protein structure capable of packaging its own genetic material, reminiscent of a virus [Butterfield et al., 2017]) demonstrates how mimicking some pathogen-like functions may be achieved without relying on pathogen-derived DNA. Nevertheless, such work falls far short of the extensive engineering required for producing a truly new viral pathogen. While packaging genetic material is one essential viral function, additional barriers exist in engineering efficient host or tissue targeting, cellular entry, genome replication, and viral particle maturation, budding, or release. Optimizing all of these functions to work effectively in concert presents an additional difficulty. Reliably engineering a brand new virus to cause specific symptoms in the host is likely to be even more challenging still. Usability as a Weapon (Medium-High Concern) The level of concern related to usability as a weapon is medium-high, primarily due to two factors. First, it may be possible to create pathogens with features not seen before. Such features could include, for example, the ability to target specific tissues or cell types using genetic logic, or the ability to produce aberrant neurological effects. Similarly, such pathogens could employ novel timing mechanisms, creating a delay between the time of exposure and the onset of symptoms. Second, in theory, pathogens designed from scratch may have a greater ability to cause harm because humans may not have been exposed to similar pathogens previously, and therefore may be immunologically naïve. Requirements of Actors (Low Concern) Design, construction, and testing of a completely novel pathogen requires capabilities that have not yet been demonstrated. While this capability is extremely broad in terms of the specific types and features of a pathogen that could be created, the high degree of expected technical PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 72 difficulty leads to an overall low level of concern in terms of the requirements of actors. Furthermore, the high uncertainty that such ambitious projects would yield the desired result in itself may lead actors away from such a path toward more reliably fruitful efforts. In general, one would expect that such ambitious, envelope-pushing projects would require well-resourced teams with deep expertise in several different technologies. A successful project would also be expected to require advanced design skills and tools, in particular software platforms that enable modeling and prediction of a pathogen’s properties, including host-pathogen interactions. Furthermore, navigating this uncharted territory would in general require many iterations of the Design-Build-Test cycle, with extensive testing needed during development. Thus, successfully designing and deploying a new pathogen would likely require a team of actors with significant time, money, and other resources to invest in the process and a permanent, well-equipped facility (as opposed to a mobile or makeshift laboratory). Potential for Mitigation (Medium-High Concern) A completely novel engineered pathogen would have the potential to frustrate existing mitigation approaches in multiple ways, leading to a medium-high level of concern for this factor. First, attempts to identify the pathogen through molecular methods—such as PCR, sequencing, or the enzyme-linked immunosorbent assay (ELISA)—would be hampered, as the pathogen would not produce results that match cleanly to known pathogens. (Indeed, in some cases one could imagine partial matches to multiple pathogens.) However, analysis of the genetic sequence of the new pathogen would likely indicate that a novel biological entity is present, providing important information. Second, symptoms of the new pathogen could mislead initial attempts at diagnosis, where common pathogens would be suspected first. Third, even if the agent is identified, correct treatment choices for the new pathogen would be uncertain. However, treatment measures taken that are common across a variety of ailments (i.e., anti-inflammatory drugs, rest, fluids) might still be germane and of some effectiveness, as such approaches are tied not just to the specific features of a given pathogen, but to general classes of symptoms in human disease (e.g., fevers, swelling, congestion, inflammation). SUMMARY • Known pathogens can be re-created. The difficulty of this re-creation increases with the size of the genome. • Engineering viruses to make them more pathogenic is possible. Design would be challenging because of knowledge limitations and because changes are generally detrimental to viruses; however, these challenges could potentially be addressed by building and testing many variations until a more pathogenic virus emerges. • Bacteria can be engineered with current technology, and the engineering of bacteria with characteristics such as multidrug resistance is an area of near- term concern. PREPUBLICATION COPY: UNCORRECTED PROOFS

73 Biodefense in the Age of Synthetic Biology • With regard to making new pathogens, the difficulty increases as the distance from natural pathogens increases. Humans have used pathogens as tools of war for centuries. Modern biotechnology has opened new opportunities for creating bioweapons, and synthetic biology further enhances and expands these opportunities. This report examined current capabilities and expected future developments related to re-creating known pathogenic viruses and bacteria, modifying existing nonpathogenic and pathogenic viruses and bacteria, and the potential creation of entirely new pathogenic agents. The possibility of re-creating known pathogenic viruses poses a relatively high level of concern. This concern is driven largely by the technical ease of synthesizing viruses (especially those with smaller genomes) and known pathogenicity of existing viruses (thus making them potentially reliable bioweapons). However, because current mitigation approaches were designed to counter natural viruses, they would be reasonably well equipped to mitigate synthetic versions of known viruses. Looking forward, it will be important to monitor technological advancements that make it easier to synthesize larger and larger viruses, which can be expected to expand the number of viruses that could be produced as bioweapons using synthetic biology. The possibility of re-creating known pathogenic bacteria poses a relatively low level of concern, largely because of the high level of technical difficulty. Because they have much larger genomes than viruses, building and booting bacteria would require a great deal of expertise, time, and resources. Given the technical difficulty of this process, actors may find it substantially easier to acquire a pathogenic bacterium through means other than synthesizing them from scratch. (In fact, the same consideration applies to viruses, even if their synthesis is easier than that of bacteria.) In addition, as with viruses, existing mitigation approaches would be expected to be reasonably well equipped to handle an attack using a synthesized known bacterial pathogen. However, two developments could increase the level of concern. If techniques using yeast were to make it far more feasible to boot synthesized bacterial genomes, or if a breakthrough makes it easier to handle large DNA fragments without shearing, the re-creation of bacterial pathogens might warrant increased concern. The use of synthetic biology to make an existing virus more dangerous poses a medium level of concern. While modifying a virus to change its phenotype may be an attractive option in theory, there are significant barriers to overcome. Such an effort would be working against finely honed virus-host dynamics evolved over millions of years, and a key factor is that modifications to a virus generally lead to attenuation. The barriers are most significant in the Design and Test phases of the Design-Build-Test cycle. While modifying a virus requires significant expertise in viral biology and challenges may be encountered in the Test phase as a result of the inability to ethically test the virus in a human, building the altered virus would be relatively straightforward. High throughput and directed evolution approaches could lower the barriers related to the Design phase. The use of synthetic biology to make an existing bacterium more dangerous poses a relatively high level of concern. This is largely driven by the technical ease of modifying bacterial genomes and the widespread availability of information about the genes involved in traits such as antibiotic resistance and toxin production. Bacteria are routinely modified for a wide variety of beneficial purposes (for example, to produce biofuels and pharmaceuticals), and the same techniques and knowledge base would likely prove useful for modifications pursued with a more nefarious intent. PREPUBLICATION COPY: UNCORRECTED PROOFS

Assessment of Concerns Related to Pathogens 74 The creation of new pathogens from scratch currently poses a relatively low level of concern, primarily because the knowledge and technologies needed to pursue such an effort are in their infancy. It is likely that a major breakthrough (or more than one) in design capabilities will be required to make this capability a reality. Relevant developments to monitor for each of these capabilities are summarized in Table 4-1. TABLE 4-1 Bottlenecks and barriers that currently constrain the capabilities considered, along with developments that could reduce these constraints in the future. NOTE: Shading indicates developments that are likely to be propelled by commercial drivers. Some approaches, such as combinatorial approaches and directed evolution, may allow bottlenecks and barriers to be widened or overcome with less explicit knowledge or tools. Capability Bottleneck or Barrier Relevant Developments to Monitor Re-creating known Booting Demonstrations of booting viruses with synthesized pathogenic viruses genomes Re-creating known DNA synthesis and Improvements in synthesis and assembly technology pathogenic bacteria assembly for handling larger DNA constructs Booting Demonstrations of booting bacteria with synthesized genomes Making existing Constraints on viral Increased knowledge of viral genome organization viruses more genome organization and/or demonstration of combinatorial approaches dangerous capable of facilitating larger-scale modifications to viral genome Engineering complex Increased knowledge of determinants of complex viral traits viral traits, as well as how to engineer pathways to produce them Making existing Engineering complex Advances in combinatorial approaches and/or bacteria more bacterial traits increased knowledge of determinants of complex dangerous bacterial traits, as well as how to engineer pathways to produce them Creating new Limited knowledge Increased knowledge of requirements for viability in pathogens regarding minimal viruses or bacteria requirements for viability (in both viruses and bacteria) PREPUBLICATION COPY: UNCORRECTED PROOFS

75 Biodefense in the Age of Synthetic Biology Constraints on viral Increased knowledge of viral genome organization genome organization and/or demonstration of combinatorial approaches capable of facilitating larger-scale modifications to viral genome PREPUBLICATION COPY: UNCORRECTED PROOFS

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Scientific advances over the past several decades have accelerated the ability to engineer existing organisms and to potentially create novel ones not found in nature. Synthetic biology, which collectively refers to concepts, approaches, and tools that enable the modification or creation of biological organisms, is being pursued overwhelmingly for beneficial purposes ranging from reducing the burden of disease to improving agricultural yields to remediating pollution. Although the contributions synthetic biology can make in these and other areas hold great promise, it is also possible to imagine malicious uses that could threaten U.S. citizens and military personnel. Making informed decisions about how to address such concerns requires a realistic assessment of the capabilities that could be misused.

Biodefense in the Age of Synthetic Biology explores and envisions potential misuses of synthetic biology. This report develops a framework to guide an assessment of the security concerns related to advances in synthetic biology, assesses the levels of concern warranted for such advances, and identifies options that could help mitigate those concerns.

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