Synthetic Biology 3.0 DNA Synthesis, Synthetic Biology, and Biosecurity

1. Synthetic Biology 3.0DNA Synthesis, Synthetic Biology, and Biosecurity Gautam Mukunda, Massachusetts Institute of Technology Department of Political Science and Program on Emerging Technologies Scott C. Mohr, Boston University Department of Chemistry and Bioinformatics Program June 25, 2007

2. SUMMARY • Introduction • Muti-Use Developments in Synthetic Biology • Security Implications of advances in DNA Synthesis and Synthetic Biology • Policy Proposals

3. DEFINITIONS Timescale • Short term: 0-5 years • Medium term: 5-10 years • Long term: >10 years Technology DNA Synthesis: de novo synthesis of 102 to 106 bp with very low error rate Synthetic Biology: The development of fundamental well-characterized parts that can be reliably combined to make devices to accomplish a particular goal

4. OFFENSIVE BIOWEAPONS PROGRAMS Suspected State Programs Russia North Korea Iran Syria China India Kazakhstan Libya Egypt Pakistan Terrorist Examples Al Qaeda laboratory in Afghanistan Aum Shinrikyo failed anthrax attack in Tokyo Anthrax attacks in the United States October 2001 Source: http://www.nti.org/ , Center for Nonproliferation Studies at the Monterey Institute of International Studies, Interviews, Koblentz: Pathogens as Weapons

5. TARGETED, INVASIN-BASED PATHOGENS (“TUMOR-EATING BACTERIA”) Species of the genus Yersina carry the inv gene that encodes an outer-membrane protein “invasin.” This gene allows bacteria to enter mammalian cells. It can be installed in E. coli and placed under control of quorum- and oxygen-sensing circuits that switch it on when the cells encounter a tumor. Adding a toxin-producing gene, suitably regulated, will af- ford a novel tumor-destroying agent. Could such an agent be re-engineered as a weapon?

6. TYPE III BACTERIAL SECRETION SYSTEMS DELIVER PROTEINS TO MAMMALIAN CELLS This astounding structure, known colloquially as “the needle,” can transfer expressed proteins to extracellular space or directly into a target cell. Many pathogens employ it already and it’s now a standard part for genetic engineers.* Could this be used with toxin genes (e.g., ricin, diphtheria) to convert an innocent commensal organism into a pathogen? *Recombinant constructs and systems for secretion of proteins via type III secretion systems, US Patent 6596509, issued to Cornell Research Foundation, 7/22/03.

7. “A TUNABLE GENETIC SWITCH FOR REGULATING MAMMALIAN GENE EXPRESSION” Work under this title was presented in a symposium on May 11 and will be published in late July. The system involved carrying genes into CHO and HEK cells with CMV and RSV vectors and regulating their expression. The separately expressed genes included EGFP, Cre recombinase, diphtheria toxin and BAX. Expression of the latter two proteins killed the cells. How much effort would be required to “weaponize” a system based on this fundamental research? 7

8. POSITIVE POTENTIAL OF SYNTHETIC BIOLOGY FOR BIOSECURITY • Develop biosensors/detectors • cf. arsenic detection system (iGEM-Edinburgh, 2006) • http://parts2.mit.edu/wiki/index.php/Edinburgh_summary_page • Design syntheses of new therapeutic agents • cf. metabolic engineering of vinblastine pathway. • http://www.synberc.org/testbeds.html • Develop mechanisms that can help cope with common pathogen-caused disease features. • cf. control ofsepsis (iGEM-Lubljana, 2006) • http://parts2.mit.edu/wiki/index.php/Ljubljana%2C_Slovenia_2006 8

9. 1. MINIMAL SHORT-TERM SECURITY IMPLICATIONS • Consensus that current and near future state of synthesis is too primitive to easily synthesize pathogens of interest • Transforming a synthesized genome into an active pathogen remains difficult and skill-intensive • Currently relatively few SB parts, and relatively few SB practitioners • Weaponizing agents is most difficult hurdle for attackers – SB unlikely to help in the short term

10. 2A. MEDIUM-TERM DEFENSIVE BENEFITS MAY BE SMALL BUT SIGNIFICANT Defensive Benefits But defenders face large obstacles Synthesis and SB are likely to produce only limited medium- term defensive benefits Better Biosensors High time pressures of reactive defense Complexity and timescales of natural biological systems Current multi-year development path of therapeutics and vaccines Production of Vaccines and Therapeutics Identification of new drug targets 10

11. 2B. MEDIUM-TERM BENEFITS TO ATTACKERS MAY BE LARGER Synthesis May Make Natural Agents Easier to Obtain SB May Make it Easier to Engineer Novel Pathogens Smallpox – probably the most dangerous natural pathogen – is currently obtainable only through theft from highly secure facilities Other natural agents (e.g. Ebola) require field biology skills that are relatively rare Most natural agents lack traits that would be useful to some attackers (e.g. contagiousness, virulence, selectivity). These traits might be engineered into them Normally harmless bacteria or viruses could be altered in order to evade detectors and defenses

12. 3. INCREASED DIFFUSION OF CAPABILITIES • Transformation of tacit to explicit knowledge is a key component of the Synthetic Biology enterprise • Removal of tacit knowledge “de-skills” the manipulation of living organisms, allowing many more people to learn to do so much more quickly • This may substantially decrease the initial investment necessary to transform a pathogen into a weapon • “De-skilling” may also flatten the capability gradient between elite and peripheral practitioners, allowing the work of the best scientists in the field to be much more easily duplicated and adapted than with conventional genetic engineering techniques

13. 4. WEAKENING OF NORMS AGAINST BW Origins of Norms Weakening in the future? Historical repugnance towards biological weapons (BW) United States and UK believed BW ineffective compared to nuclear weapons US unilaterally renounced offensive BW in 1969 Continued non-use of weapons reinforces taboo against them SB may make BW more effective, increasing the temptation to use them Increased perceived threat may make states conduct aggressive defensive programs indistinguishable to outsiders from offensive programs

14. 5. SUBSTANTIALLY INCREASED UNCERTAINTIES • Traditional genetic engineering techniques have difficulty utilizing discoveries from the rest of biology • SB, with its focus on chassis and parts that are easily assembled, is meant to be able to do so much more rapidly • Thus several discoveries from unrelated fields could combine in inherently unpredictable ways (immune-invisible chassis + sleep-inducing hormones + reliable penetration of blood-brain barrier = non-lethal biological weapon)

15. CREATE A SURVEILLANCE REGIME High Economies of Scale Low Economies of Scale Centralized synthesis Software monitoring of requested sequences Database of requested sequences Prize system to discover security loopholes Focused attention on independent syntheses Norm of peer monitoring Distributed synthesis Potential “Garage biohacker” culture Licensing of powerful synthesizers? Required inspection of powerful synthesizers? Norm of peer monitoring

16. STRENGTHEN NORMS AND COMMUNITY Community Responses Establish a funded Synthetic Biology Professional Organization “Safety hold” on experiments Inculcate norm of monitoring activities of other researchers for potential misuse Create back-channel contacts between SB researchers in different countries Policy Responses Commitment to open biodefense research De-classify as much research as possible All BL4 labs on open facilities Extend BWC to make research, production, and use of BW a crime under international criminal law

17. PURSUE DEFENSIVE OPPORTUNITIES • Synthesis and SB may allow faster and more flexible development of biodefenses • Such developments are likely to rely on significant discoveries from outside SB (e.g. immunology) • Research is inhibited by disconnect between academic programs and needs of governments and pharmaceutical companies The long-term revolutionary potential of SB should be fully exploited Create committee from leaders in SB, pharmaceutical companies, governments, and public health with funding from governments and industry Mandate to fund highly speculative research taking advantage of potential of SB

18. ACKNOWLEDGEMENTS Rocco Cassagrande George Church Jim Collins Drew Endy Jay Keasling Tom Knight Gregory D. Koblentz Kenneth Oye Randy Rettberg Partial funding provided by: The National Science Foundation The Paul & Daisy Soros Fellowships for New Americans 18

19. APPENDIX

20. CDC Select Agents* – Bacteria • Bacillus anthracis (spores) • Brucella abortus • Brucella melitensis • Brucella suis • Burkholderia mallei (aka Pseudomonas mallei) • Burkholderia pseudomallei (aka Pseudomonas pseudomallei) • Clostridium (botulinum- producing species) • Coxiella burnetii • Francisella tularensis • Rickettsia prowazekii • Rickettsia rickettsii • Yersinia pestis n = 12 * Not including agents listed only by USDA.

21. CDC Select Agents* – Fungi • Coccidiodies immitis • Coccidiodies posadasii n = 2 * Not including agents only on USDA lists.

22. CDC Select Agents* – Viruses I • Central European Tick-borne encephalitis • Cercopithecine herpesvirus 1 • Crimean-Congo haemorrhagic fever • Eastern Equine encephalitis • Ebola • Far Eastern Tick-borne encephalitis • Flexal South American haemorrhagic fever • Guanarito South American haemorrhagic fever • Hendra • Junin South American haemorrhagic fever • Kyasanur Forest disease • Lassa fever • Marburg * Not including agents only on USDA lists.

23. CDC Select Agents* – Viruses II • Machupo South American haemorrhagic fever • Monkeypox • Nipah • Omsk haemorrhagic fever • Reconstructed 1918 influenza • Rift Valley fever • Russian Spring and Summer encephalitis • Sabia South American haemorrhagic fever • Variola major (smallpox) • Variola minor (alastrim) • Venezuelan Equine encephalitis n = 24 * Not including agents only on USDA lists.

24. CDC Select Agents* – Toxins • Abrin • Botulinum neurotoxins • Clostridium perfingens epsilon toxin • Conotoxins • Diacetoxyscirpenol • Ricin • Saxitoxin • Shiga-like ribosome-inactivating proteins • Shigatoxin • Staphylococcal enterotoxins • Tetrodotoxin • T-2 toxin n = 12 * Not including agents only on USDA lists.

25. CDC Select Agents – Nucleic Acids n = 0 !

26. Additional Potential Bioterrorism Agents • Chlamydia psittaci • Cryptosporidium parvum • Escherichia coli O157:H7 • hantavirus • Salmonella species • Shigella species • Vibria cholerae