Addressing Future Challenges to the Biological and Toxin Weapons Convention in Connection with Scientific and Technological Developments Frank Notka -

Addressing Future Challenges to the Biological and Toxin Weapons Convention in Connection with Scientific and Technological Developments Frank Notka - March 6th, Technical Workshop, Stockholm

The relevance and potential applicability of

screening framework guidance for commercial

providers of synthetic double-stranded DNA:

Current software screening and review

mechanisms

Company factsEmployees ~ 190 employees

Locations Headquarters: Regensburg, Germany

Business areas Life science, Synthetic Biology; research, development

Status Worldwide leading provider of gene synthesis

Affiliate of Life Technologies since Dec. 2010

Capacity 4 Mbp/month (2010) ~ E.coli genome; 80,000 constructs in 2010; To Date: More than 180,000 individual gene constructs built

Quality DIN ISO 9001:2008 certified

Customers base Top 20 pharmaceutical, chemical companies, biotech industry as well as universities and research institutes

IGSC was founded by:

Division: Synthetic BiologyDivision: Synthetic Biology

Charter members

http://www.genesynthesisconsortium.org

Accidental exposure to a pathogen or toxin could adversely affect:

Laboratory workers General public Plants and animals Environment

Deliberate misuse of technology to cause harm to: Humans Plants and animals Environment

Major Categories of Risk Posed by Biotechnology Research

Biosafety Biosecurity

BIOHAZARDBIOHAZARD

oppo

rtunit

y

Balanced Discussion to Take Advantage of the Opportunities and Minimize Risks

IGSC Goals and Objectives

1) To design and apply a common Protocol to screen both the sequences of synthetic gene orders, and the customers who place them.

2) To work together with governments and others concerned to promote the beneficial application of gene synthesis technology, and to safeguard biosecurity.

The IGSC harmonized screening protocol is:

• In line with the draft guidelines

• Based on experience and existing screening processes of founding members

• Compulsory for IGSC members

• Publically available*

*http://www.genesynthesisconsortium.org/

IGSC Goals and Objectives

1) To design and apply a common Protocol to screen both the sequences of synthetic gene orders, and the customers who place them.

2) To work together with governments and others concerned to promote the beneficial application of gene synthesis technology, and to safeguard biosecurity.

Synthetic biology: key events and considerations

2000 2010

USG dual use research

policy + rDNAguidelines

revision

USG dual use research

policy + rDNAguidelines

revision

Syn Bio 1.0 @ MIT

Syn Bio 1.0 @ MIT

NSABB Reports on Synthetic Genomics, Oversight Framework,

Synthetic Biology, 2006-2010





Human Genome Project, 2003

Human Genome Project, 2003

2007-2009Genomic transplantation,

assembly of whole bacterial genome, interspecies genomic

transfer



transfer



transfer

Artemisin, 2006

In vitro synthesis of polio virus, 2002 BioBricksTM Artemisin, 2006Artemisin, 2006

In vitro synthesis of polio virus, 2002

In vitro synthesis of polio virus, 2002 BioBricksTMBioBricksTM

2003

NSABB establishment

200420032003

NSABB establishment

2004

NSABB establishment

2004

2000Gene

Synthesis GenomeSynthesis

ATG ATC TGT CAC GCA GAG CTAATG ATC TGT CAC GCA GAG CTA

2009

FBI Reporting

Program

2010

Presidential

commission

2009

Screening

framework

Screening Framework Guidance, Oct 2010

Department of Health and Human ServicesSCREENING FRAMEWORK GUIDANCE OR PROVIDERS OF SYNTHETIC DOUBLE-STRANDED DNA

Following these guidance is voluntary

The cornerstones:• Screening of sequences• Screening of customer• Follow-up screening if above raises concerns• Proper record retention• Recommendations regarding screening software

The Guidance was developed, in light of providers’ existing protocols, to be implemented without unnecessary cost and to be globally extensible, both for U.S.-based providers operating abroad and for international providers

Providers of synthetic dsDNA have two overriding responsibilities in this context: • Providers should know to whom they are distributing a product • Providers should know if the product that they are synthesizing and distributing contains, in part or in

whole, a “sequence of concern”

Screening Framework Guidance, Summary

Screening Practice, Australia Group

an informal forum of member countries with the objective to ensure that exports do not contribute to the development of chemical or biological weapons, and to strengthen global security through harmonization of export controls (http://www.australiagroup.net/en/index.html).

Ratified in member countries’ export control lists

http://www.australiagroup.net/en/index.html



Screening Practice, Australia Group Definitions

Genetic Elements and Genetically-modified Organisms:

•that contain nucleic acid sequences associated with the pathogenicity of any of the

microorganisms in the list.

•that contain nucleic acid sequences coding for any of the toxins in the list, or for their sub-

units.

Technical note:

•Genetic elements include inter alia chromosomes, genomes, plasmids, transposons, and

vectors whether genetically modified or unmodified.

•Nucleic acid sequences associated with the pathogenicity of any of the micro-organisms in

the list means any sequence specific to the relevant listed micro-organism:

• that in itself or through its transcribed or translated products represents a significant

hazard to human, animal or plant health; or

• that is known to enhance the ability of a listed micro-organism, or any other organism

into which it may be inserted or otherwise integrated, to cause serious harm to human,

animal or plant health.

Screening practice @ Life Tech

www

BL

AS

T

Sequence HostSequence Function

?? ok

Country checkCustomer check

checkFedEx

critical sequence list(AG list, CDC)

A

Mok

A: automated

M: manual

ok?

(9 PhDs) molecular biologists

Sales Team(17)

ok?

Sequence check

BioSafety BioSecurity

atlas

„Gene Technology Law“

Customs regulationExport controlDocumentationSurprise visit

Sequence Evaluation Process – WORK FLOW

Work & information flow

Rules:

CP

M (

9 P

hDs)

Sequence input

Biosecurityissues?

Unknown sequenceartificial sequences

Proceed with order (quote)

Order placed (customer)

Docu-request & quote AWPC EVE BAFA

Order placed (customer)

Documents provided?

Start synthesis

Contactcustomer

no

no

Screen sequence- Internal Black list (taxID & seqID, incl. IGSC db)- Internal White list (taxID based)

Blast sequence- NCBI BLASTX, BLASTN or BLASTP- Results: seqID (gi) and taxID

match?

no

unsorted 6-frame translation, using standard blast parameter, limit: 44 nt

every result produced by the blast is considered, independently from the query coverage (homology)

gi = identified sequence over-rules taxID = identified organism

Identify a gene from a requested

sequence

Automated

Evaluate dual-use potential

Automated plus Expert

approval

Follow-up customer/

authorities

Manual

Sequence Evaluation Process – Examples

EU AG non-AGgreenyellow

red

OK OK OKOK CD EPCD EP EP

Shipping addressBiosecurity evaluation result

AG: Australia group

CD: Customer document

EP: Export control

Origin Gene Shipping address BS level Export

Foot-and-mouth disease virus

130 aa from polyprotein USA - AG red Permission req.

Coxiella burnetii (Rickettsiae)

isocitrate dehydrogenase

India – non AG yellow Permission req.

Coxiella burnetii (Rickettsiae)

isocitrate dehydrogenase

Italy - EU yellow yes

Ebola virus Envelop protein Italy - EU red Customer document

Ebola virus Envelop protein Switzerland - AG red Permission req.

Ebola virus Matrix protein Switzerland - AG yellow Customer document

Internationalization & screening framework guidance

• Can deliver botulinus toxin genes within Europe, but cannot send Dengue Env to Novartis Switzerland or NIH US

− What is the consequence of a „hit“ for shipment in Europe?

− How can export limitations to institutions such as e.g. delivering genes from EU to NIH be overcome (e.g. for the case of an epidemic swine flue vaccine)?

• Relevance and need for guidance− Central role of customer screen and customer legitimacy− Definition of “sequence of concern”− Export regulations

Identified gaps / open questions

Technical advising from experts – defining sequences of concern

• Uniform screening practice / ~criteria

• Australian Group List names only organisms, not sequences

• Definition of „genes associated with pathogenicity“ leaves room for interpretation

• Definition of “Match” is central for sequence evaluation

A screening database that is continually updated

• Ideally maintained by the U.S. Government / EU and international organizations

• Most complete, updated & classified

Internationally harmonized list of suspicious persons & organizations

• User certification (?)

What’s Next?

• Improve sequence screening methods and tools (data base, taxID- and gi-based)

• Incorporation of U.S. Screening Guidance into Protocol

• Participate in policy development and resource discussion

− Centrally curated sequence database for screening?

− Regulate access to screening data base?

− Funding support for screening software?

− Consider moving toward certification for Federal Guidance compliance

Ways to bypass US/EU based Gene Synthesis companies

• Can order oligonucleotides @ oligo firms

• Synthesize oligonucleotides on purchased synthesizer (new or used) or on synthesizer that was built according to construction plans available via internet

• Oligos assembled via published technologies

• Synthetic genes ordered from Non-US / Non-EU based companies

• Use conventional genetic engineering

• Isolate harmful species from natural habitats and cultivate

• Customer screen / identification as important as sequence screening

Conclusion

• Delivery of sequences of concern to customers is regulated by national export law according to respective lists: e.g. Export control list, CDC list, selected agents list (genes and organism), K-List (countries) and Haddex List (institutions/companies)

• An internationally harmonized screening framework guidance is appreciated, but should be developed in close collaboration with commercial gene synthesis providers

• The screening practice of the large commercial gene synthesis providers is in accordance with the current US screening framework guidance

• In addition to regulation, governments should provide technical specifications (sequences, blast parameter, update process, etc)

Thank you for your attention