Emerging Dual-Use Technologies in the Life Sciences

Challenges and Policy Recommendations on Export Control

Dr Mirko HimmelEU Export Control Forum

Brussels, 13 December 2019

Emerging Technologies: A Scientist‘s View

• Term „emerging technology“ not clearly defined!

• Emerging technologies are ...- “new“?- “novel“?- “not yet widespread in use in a particular scientific field“?- “somewhat risky?”

• How to identify an “emerging technology” in the life sciences? Does this technology fall under export control measures?

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Emerging Technologies: Definitions

• Emerging technologies are often described as technologies that have disruptive potential but have not yet been developed to their fullest potential (see Brockmann et al., 2019).

• Emerging technologies may have great strategic value and the potential to be adopted for important military and non-military industrial purposes(see Brockmann, 2018).

• At least some emerging technologies may pose a risk to national securityand fall within the scope of international arms control treaties and non-proliferation measures (see Himmel, 2019).

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Emerging Technologies: Attributes

Attributes of an “emerging technology” (Rotolo et al., 2015):

Radical novelty: May appear either in the method or the function of the technology

Relatively fast growth: A technology may grow rapidly in comparison with other technologies in the same domain(s)

Coherence: Attributes of a technology such as 'sticking together', 'being united', 'logical interconnection' and 'congruity’ (e.g., unified technical terms get established)

Prominent impact: Technological achievements, socio-cultural, securityUncertainty and ambiguity: Lack of knowledge of possible outcomes such as

unintended/undesirable consequences deriving from the (potentially uncontrolled) use of the technology

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Emerging Technologies in the Life Sciences

• Genome editing (genetic engineering technology)

• Synthetic biology

• Nanobiotechnology

• Digital biological data acquisition and processing

• Additive manufacturing/3D printing

(Source: www.arl.army.mil)

that are complementary to a linker sequenceattached to the intended cargo (Fig. 1D). Afternanorobot folding and purification, cargo loadingwas carried out by adding linker-modified pay-load in molar excess to attachment sites and in-cubating at room temperature for 12 hours. Twotypes of cargo were loaded: 5-nm gold nano-particles covalently attached to 5′-thiol–modifiedlinkers (18), and various Fab´ antibody fragmentsthat were covalently attached to 5′-amine–modified linkers using a HyNic/4FB couplingkit (Solulink, San Diego, California). We usednegative-stain transmission electron microscopy(TEM) to analyze the device in closed and openstates, with and without cargo (Fig. 1F). We ob-served by manual counting that on average fourattachment sites were populated when loadinggold nanoparticles, and three sites were populatedwhen loading antibody fragments (fig. S11).

One obstacle to overcome in constructing a“spring-loaded” device was to ensure assemblyto high yield in its closed state. Like hands thatset a mousetrap, two “guide” staples were in-

corporated adjacent to the lock sites that span thetop and bottom domains of the device (Fig. 1E).The guide staples include 8-base toehold over-hangs and could be removed after folding andpurification steps by adding a 10:1 excess of fullycomplementary strands to the mixture (19). Weobserved that foldingwith the aid of guide staplesincreased the yield of closed robots from 48%to 97.5%, as assessed by manual counting ofnanorobots images by TEM (fig. S17).

To examine nanorobot function, we selected apayload such that robot activation would be cou-pled to labeling of an activating cell (Fig. 2A).Robots loaded with fluorescently labeled anti-body fragments against human leukocyte antigen(HLA)–A/B/Cwere mixed with different cell typesexpressing humanHLA-A/B/C and various “key”combinations (described below) and were ana-lyzed by flow cytometry. In the absence of thecorrect combination of keys, the robot remainedinactive. In the inactive state, the sequesteredantibody fragments were not able to bind the cellsurface, resulting in a baseline fluorescence sig-

nal. However, when the robot encountered theproper combination of antigen keys, it was freedto open and bind to the cell surface via its anti-body payload, causing an increase in fluorescence.We used key-neutralizing antibodies in compe-titive inhibition control experiments to verify thatnanorobots were not activated by a non–ligand-based mechanism (fig. S25).

The robot could be programmed to activate inresponse to a single type of key by using thesame aptamer sequence in both lock sites. Al-ternatively, different aptamer sequences could beencoded in the locks to recognize two inputs.Both locks needed to be opened simultaneouslyto activate the robot. The robot remained inactivewhen only one of the two locks was opened. Thelock mechanism is thus equivalent to a logicalAND gate, with possible inputs of cell surfaceantigens not binding or binding (0 or 1, respec-tively) to aptamer locks, and possible outputs ofremaining closed or a conformational rearrange-ment to expose the payload (0 or 1, respectively)(Fig. 2B).

FA C+ +

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Fig. 1. Design and TEM analysis of aptamer-gated DNA nanorobot. (A) Sche-matic front orthographic view of closed nanorobot loaded with a protein pay-load. Two DNA-aptamer locks fasten the front of the device on the left (boxed)and right. (B) Aptamer lock mechanism, consisting of a DNA aptamer (blue)and a partially complementary strand (orange). The lock can be stabilized in adissociated state by its antigen key (red). Unless otherwise noted, the lockduplex length is 24 bp, with an 18- to 24-base thymine spacer in the nonaptamerstrand. (C) Perspective view of nanorobot opened by protein displacement ofaptamer locks. The two domains (blue and orange) are constrained in the rear by

scaffold hinges. (D) Payloads such as gold nanoparticles (gold) and antibody Fab´fragments (magenta) can be loaded inside the nanorobot. (E) Front and sideviews show guide staples (red) bearing 8-base toeholds aid assembly of nano-robot to 97.5% yield in closed state as assessed by manual counting. Afterfolding, guide staples are removed by addition of fully complementary oligos(black). Nanorobots can be subsequently activated by interaction with antigenkeys (red). (F) TEM images of robots in closed and open conformations. Leftcolumn, unloaded; center column, robots loaded with 5-nm gold nanoparticles;right column, robots loaded with Fab´ fragments. Scale bars, 20 nm.

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5For further reference see: National Academies of Sciences, 2018; Kirkpatrick et al., 2018

Genome Editing: A New Era?

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Emerging Technologies: Genome Editing

• Genome editing: targeted manipulation of genomes of cells or whole organisms by genetic engineering techniques

• CRISPR/Cas9 genome editing tool-set as most promising candidate (key molecular components derived from nature!)

• Possible applications include generation of optimised crops & livestock; cure of genetic diseases; generation of optimised microorganisms for biotechnological production processes; building of gene drives for the eradication of infectious diseases

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Emerging Technologies: Genome Editing

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Doudna et Charpentier, Science, 2014

Genome Editing: How to access risk potentials?

• Genome editing is an emerging technology which full range of applications and risks for human/environmental safety and security is still not known.

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type II, IV, V, and VI CRISPR systems49. Although researchersrepurposed many different CRISPR/Cas systems for genometargeting, the most widely used one is the type II CRISPR-Cas9system from Streptococcus pyogenes. Because of the simple NGGPAM sequence requirements, S. pyogenes’ Cas9 (spCas9) is usedin many different applications. However, researchers are stillactively exploring other CRISPR systems to identify Cas9-likeeffector proteins that may have differences in their sizes, PAMrequirements, and substrate preferences. In the last few years,more than 10 different CRISPR/Cas proteins have been repur-posed for genome editing (Table 1). Among these, some of therecently discovered ones, such as Cpf1 proteins from Acid-aminococcus sp (AsCpf1) and Lachnospiraceae bacterium(LbCpf1), are particularly interesting50–52. In contrast to thenative Cas9, which requires two separate short RNAs, Cpf1naturally requires one sgRNA. Furthermore, it cuts DNA at targetsites 3′ downstream of the PAM sequence in a staggering fashion,generating a 5′ overhang rather than producing blunt ends likeCas9 (Table 1).

Naturally found Cas9 variants are large proteins, which addsparticular limitation when it comes to their packaging anddelivery into different cell types via Lenti or Adeno Associatedviruses (AAV). For example, the widely used SpCas9 protein is

1,366 aa, which creates a particular therapeutic delivery challengedue to the limited packaging capacity of AAV. Thus, smaller Cas9variants have greater therapeutic potential. To this end, the dis-coveries of 1082 aa Cas9 from Neisseria meningitides (NmCas9)53, 1053 aa Cas9 from Staphylococcus aureus (SaCas9)54,55, and984 aa Cas9 from Campylobacter jejuni (CjCas9)56 are majorforward steps toward this goal. However, the tradeoff is that thesesmaller Cas9 proteins require more complex PAM sequences. TheSaCas9 requires a 5′-NNGRRT-3′ PAM sequence54,55,57 whereasCjCas9 requires a 5′-NNNNACAC-3′ PAM sequence56. There-fore, these smaller Cas9 proteins have relatively limited targetingscope and flexibility in genome targeting compared to SpCas9despite the reduction in size.

Re-engineering CRISPR-Cas9 toolsExploring different CRISPR systems requires extensive under-standing and characterization of new Cas proteins. Thus, inparallel to these studies, there are increasing efforts to re-engineerthe already well-characterized Cas9 proteins. This researchdirection is focusing on achieving three major goals: (i) reducingthe size of Cas9 nucleases, (ii) increasing their fidelity, and (iii)expanding the targeting scope of Cas9 variants. Although therehas been a limited advance in reducing the size of existing Cas9proteins, several groups have altered the Cas9 PAM requirementsand targeting specificity. In one such study, researchers used anunbiased selection strategy to identify variants of SpCas9 andSaCas9 with more relaxed PAM sequence requirements58,59. Inline with these findings, a different study utilized a structure-guided design strategy to re-engineer FnCas9 to recognize YGPAM sequences instead of NGG60.

In addition to these studies that expand the targeting scope ofCRISPR tools, researchers are actively developing novel ways toincrease the targeting specificity of the CRISPR-Cas9 system.Understanding the extent of off-target effects of CRISPR-Cas9targeting has been one major goal. Given that CRISPR systemshave evolved as a defense system against viruses that tend tofrequently mutate, a slightly less specific CRISPR system would beadvantageous to bacteria. Indeed, the early efforts to understandCRISPR targeting specificity highlighted this fact and demon-strated that the system may potentially have off-target effects61–65. In addition to these initial studies, researchers utilized alter-native genome-wide tools to understand CRISPR-Cas9 targetingspecificity. To this end, we and others have used the chromatinimmunoprecipitation and high throughput sequencing (ChIP-Seq) approach to map DNA binding sites of catalytically inactiveSpCas9 in vivo66,67. These whole-genome mapping studies

Table 1 Naturally occurring major CRISPR-Cas enzymes

Size PAM sequence Size of sgRNA guiding sequence Cutting site Reference

spCas9 1368 NGG 20 bp ~ 3 bp 5′ of PAM Jinek et al.42

Gasiunas et al.43

FnCas9 1629 NGG 20 bp ~ 3 pb 5′ of PAM Hirano et al.60

SaCas9 1053 NNGR RT 21 bp ~ 3 pb 5′ of PAM Mojica et al.57

NmCas9 1082 NNNNG ATT 24 bp ~ 3 bp 5′ of PAM Hou et al.53

St1Cas9 1121 NNAGA AW 20 bp ~ 3 bp 5′ of PAM Gasiunas et al.43

Cong et al.45

St3Cas9 1409 NGGNG 20 bp ~ 3 bp 5′ of PAM Gasiunas et al.43

Cong et al.45

CjCas9 984 NNNNACAC 22 bp ~ 3 bp 5′ of PAM Kim et al.56

AsCPf1 1307 TTTV 24 bp 19/24 bp 3′ of PAM Yamano et al.50

Kim et al. 2016LbCpf1 1228 TTTV 24 bp 19/24 bp 3′ of PAM Yamano et al.50

Kim et al. 2016Cas13 Multiple orthologs RNA targeting 28 bp Abudayyeh et al. 2017

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Fig. 2 CRISPR-based genome-targeting tools are widely used. Number ofPubMed publications over the last 12 years that had the word “CRISPR” or“Cas9” in the abstract or title. **Number of publications in 2018 is projectedto be more than 5000

REVIEW ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04252-2

4 NATURE COMMUNICATIONS | �(2018)�9:1911� | DOI: 10.1038/s41467-018-04252-2 | www.nature.com/naturecommunications

Growing number of published studies using CRISPR/Cas9

** >5,000 paper

Adli, M. (2018). The CRISPR tool kit for genome editing and beyond. Nat Commun, 9(1):1911.

MIT Technology Review (Feb. 2016):

Genome editing is a weapon of mass destruction.

That’s according to James Clapper, U.S.director of national intelligence, who onTuesday, in the annual worldwide threatassessment report of the U.S. intelligencecommunity, added gene editing to a list ofthreats posed by “weapons of massdestruction and proliferation.”

„CRISPR—a weapon of mass destruction?“Science. 2016

Genome Editing: A Security Threat?

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Challenges for Export Control Regimes

• Disciplinary fragmentation & growing body of scientific literatureØ Increasingly complex to identify important actors working on sensitive research topics; each

year ~2.5 million scientific papers are published worldwide > unknown number of papers including dual-use research of concern > review? oversight?

• Convergence of biology and chemistryØNew biotechnological production processes could be misapplied to the production of highly

toxic chemicals, bioregulators or toxins.

• Transfer of intangible technologies & Legal harmonizationØProtocols/methods descriptions could include sensitive information;

• Circumvention of export controls & Non-conventional trade flowsØ Illicit trafficking of dual-use items through non-conventional trade flows organized over the

darknet? Cloud-based laboratories and biofoundries offer remote access to laboratory equipment crossing national borders > Blueprints? Fragmented processing orders below triggers? 11

Recommendations for Export Control

• Develop a systematic science and technology review processØEU-specific review mechanism for export control of dual-use items in the life sciences & risk

assessment framework for emerging technologies; Establish EU Export Control Support Unit?

• Harmonize key technical termsØA revised definition of dual-use items in the life sciences could go beyond conventional military

and state-centric approaches to security and could explicitly mention broader security implications.

• Increase awareness of export control measures in academia & industryØHarmonized education and training courses for various stakeholder groups involved in dual-use

activities in the life sciences, governmental research institutions, industry and the DIY/open science community

• Include academia in the e-Licencing processØEnables reduction in the bureaucratic burden (e.g. electronic exchange of scientific and

technical information!); provides guidance for exporters & end-users (see MTAs) 12

Conclusion

• Unified classification criteria are needed for identifying relevant emerging dual-use technologies in the life sciences for the purpose of export control.

• Raising awareness among researchers working in biotechnology industries and the life sciences is a key element of strengthening the EU export control system.

• Export control measures must be designed to prevent the proliferation of dual-use technologies but also to back freedom of research wherever possible.

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T H A N K Y O U !

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References

• Adli, M. (2018). The CRISPR tool kit for genome editing and beyond. Nat Commun, 9(1):1911.

• Brockmann, K., Bauer, S. and Boulanin, V., ‘BIO PLUS X: Arms Control and the Convergence of Biology and Emerging Technologies’, (SIPRI: Stockholm, 2019), p. 2.

• Brockmann, K., ‘Drafting, implementing and complying with export controls: The challenge presented by emerging technologies’, Strategic Trade Review, vol. 8, no. 6 (2018), pp. 5–28.

• Doudna, J. A. and Charpentier, E. (2014). Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213):1258096.

• Himmel, M. (2019). Emerging Dual-Use Technologies in the Life Sciences: Challenges and Policy Recommendations on Export Control. Non-Proliferation and Disarmament Papers, (64).

• Kirkpatrick, J., Koblentz, G. D., Palmer, M. J., Perello, E., Relman, D. A., & Denton, S. W. (2018). Editing Biosecurity: Needs and Strategies for Governing Genome Editing. George Mason University.

• National Academies of Sciences, Engineering, and Medicine. (2018). Biodefense in the age of synthetic biology. National Academies Press.

• Rotolo, D., Hicks, D., and Martin, B. R. (2015). What is an emerging technology? Research Policy, 44(10):1827–1843.

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3D Printing: Production of Bioreactors

Charbe, N., McCarron, P. A., and Tambuwala, M. M. (2017). Three-dimensional bio- printing: A new frontier in oncology research. World J Clin Oncol, 8(1):21–36.

Sugar cube-sized 3-D-printed gel lattices containing yeast can ferment glucose into ethanol for days, potentially bringing down the cost of industrial fermentation. Source: c&en News, 2018

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CyberbiosecurityPeccoud, J., Gallegos, J. E., Murch, R., Buchholz, W. G., and Raman, S. (2018). Cyberbiosecurity: From naive trust to risk awareness. Trends Biotechnol, 36(1):4–7.

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Gene Drive TechnologyArtificially introduced gene drive self-integrates into wildtype chromosome within one individual organism

Gene drives bias inheritance of selected alleles

Future gene drive applications

Esvelt, K. M., Smidler, A. L., Catteruccia, F., and Church, G. M. (2014). Emerging Technology: Concerning RNA-guided gene drives for the alteration of wild populations. eLife, 3:e03401.