Commercial Gene Synthesis Technology John Mulligan Berkley Synthetic

Commercial Gene Synthesis Technology John Mulligan Berkley Synthetic Biology Class, April 5, 2006

Topics Ø Commercial gene synthesis today Ø Issues and technology for the future Ø Governance and the potential for nefarious applications of synthetic biology

Access to DNA is Central to Modern Biology Ø Biomedical Research Ø Biology Ø Agriculture Ø New areas such as Synthetic Biology

Acquiring and Modifying DNA is Costly Ø Researchers spend > $800 MM/year on reagents to clone and modify genes • Deutsche Banc Alex Brown, 2000 Ø Every $1 spent on reagents represents an additional $2 to $5 of fully loaded costs • Labor, overhead, facilities, etc. Ø Billions of dollars in time and effort every year • Roughly $1 billion in direct costs to NIH • $1 -$2 billion to industry

Gene Synthesis Improves Research Productivity Ø Less costly than other methods for many projects today ($1. 25 to $1. 60 per base pair today) • Industrial groups believe their internal costs with other methods to be ~$2 per bp • Academic groups have lower costs but still find synthesis economical for many projects • Cost of synthesis continues to decline rapidly Ø Complete control of sequence allows improved experimental design and new experimental approaches • Use the perfect gene for your experiment instead of the gene you have in the freezer

Commercial Gene Synthesis Ø Potentially a substitute for $1 to $2 billion in fully loaded costs Ø We estimate the current market is $20 million to $30 million a year • Revenues rowing at 30% to 50% a year - Volume growth much higher • Highly fragmented: 50 or more companies in this area world wide Ø Still a tiny fraction of the overall molecular biology market • We expect it to grow rapidly but to take 5 -10 years to reach a significant fraction of the molecular biology market

Blue Heron Order Mix Ø Standard Orders • • • $500 to $50, 000 One to ~50 genes, as fast as possible Standard delivery schedule Ø High-Volume, Time-Sensitive (Corporate) • • • Hundreds of kilobases, as fast as possible Negotiated delivery schedule A 200 kb project in 2004 and a 450 kb project in 2005 Ø High-Volume, Time-Insensitive (Government) • • • Thousands of kilobases Extended Delivery, discounted price Enables full capacity utilization to leverage fixed costs and maximize economies of scale

Gene Synthesis Technology Ø In use since the late ’ 70’s but only beginning to be widely used Ø Challenges • • • Error rate: ~1/300 Mismatched hybridization can lead to scrambled order Reliability impacts speed and cost Ø Three general approaches • • • “One pot” ligation and/or PCR Convergent assembly Solid phase assembly

PCR Assembly Multiple oligonucleotides in a single reaction. PCR Amplification

PCR Assembly Ø Simple to do, often works • The technology used by nearly all commercial providers • Many published protocols Ø Limitations • Some sequences are difficult or impossible to PCR • Difficult sequences can add to the cost and delivery time

Convergent Assembly A series ligation and purification steps, each involving only two fragments.

Convergent Assembly Ø A series of simple, reliable reactions Ø Works on almost any sequence Ø But, it is slow and more expensive than PCRbased methods for many genes

Solid-Phase Assembly Within each column, double-stranded oligos (duplexes) are sequentially added to a solid support, with intervening wash steps. Inside column

Solid-Phase Assembly Attach duplex to solid phase support Inside column

Solid-Phase Assembly Wash Inside column

Solid-Phase Assembly Add the second duplex into column Inside column

Solid-Phase Assembly Attach the second duplex to the first duplex Inside column

Solid-Phase Assembly Wash Inside column

Solid-Phase Assembly Add the third duplex into column Inside column

Solid-Phase Assembly Attach the third duplex Inside column

Solid-Phase Assembly Repeat to assemble complete fragments and elute from column Inside column

Solid-Phase Assembly Ø Simple reaction: two fragments, three ends Ø Drive reaction with molar excess Ø Wash away side reactions Ø Fully automated at Blue Heron

Solid Phase Assembly of Whole Genes Oligos Subtarget Cloned Sequence verified Restricted Subtargets Final Clone

Gene. Maker® Overview Ø Proprietary software designs build strategy Ø Oracle database instructs instruments to build Ø Oligos are synthesized and hybridized Ø Patent-pending automated solid phase assembly Ø Cloning and sequencing Ø Error removal methods throughout process

Issues and Technology for the Future

Gene Synthesis is Complex Ø Every order is different • Every gene is made from a dozen to several thousand parts Ø Every part is new and used for only one order Ø The smallest parts are chemicals • Mixed populations of good and bad parts • Error rate of on in a few hundred Ø Larger parts are biological • Unpredictable behavior Ø The final product must be perfect

Existing Manufacturing Tools are Inadequate Ø Commodity market • Prices drop 30% to 50% / year • We must drop production costs at least this fast Ø Mass customization used in some industries • Have not found one where every part is new Ø Handling high failure rates is critical to controlling manufacturing costs Ø Existing tools focused on assembly-line production, “job shops”, custom engineering • None

Automated Laboratory vs. Manufacturing Ø Most or all gene synthesis today is carried out in sophisticated laboratories with some automation • Ph. Ds involved • Difficult to scale rapidly Ø Within a few years, nearly all commercial gene synthesis will be carried out in manufacturing facilities • • Largely automated Robots for production People for process development Highly sophisticated process control and scheduling Ø Interesting, meaty problems for operations research…

Blue Heron is a Software Company Ø Integrated manufacturing system • • Automated storage Integrated materials handling: e. g. , robot arm on a rail Off the shelf components: pipettors and incubators Proprietary process Ø Lots of software • Automated design of manufacturing process • Database control to track every fragment and manage rework cycles • Sophisticated scheduling • Integration software • Protocol software on individual instruments

Nefarious Applications and Governance

The Potential for Biowarfare Applications Ø Many researchers synthesize or clone pathogenic DNA as they work to understand the basic biology of the pathogen and to develop new therapeutics Ø Most viral genomes are within the range of today’s technology • Blue Heron delivered the fragments for a >25 kb virus in 2004 • Vaccinia is 180 kb - ould be done in 6 -12 months, 40 SNPs Ø One or more bacterial genomes will be synthesized within the next year Ø Nefarious uses of synthesis are possible

Gene Synthesis Technology is Widespread Bioneer Corporation 49 -3, Munpyeong-dong, Daedeok-gu, Daejeon 306 -220, Korea “The capacity of this facility is to produce 7. 2 tons of phosphoramidite per year… Currently we have (the) capacity of producing 20, 000 oligos per day… Bioneer offers a special gene synthesis service. ” But the vast majority of the sophisticated molecular biology capacity is in Europe and North America

Controlling Synthesis Technology is Difficult Ø Synthesis materials are easy to acquire • Any sophisticated chemistry group could build oligonucleotide synthesis capacity from scratch • For large-scale synthesis groups the “drop at the bottom of a reagent bottle” can add up to kilograms of phosphoramidite per year- tracking the materials is not feasible Ø PCR-based synthesis works on many sequences Ø Transforming and growing bacteria is low-tech

New Methods Extend Synthesis Capabilities Build genes with a modified ink-jet printer?

Garage Technology in Five Years? Ø Lone hackers with few resources NO Ø Governments or organizations YES • Any country or moderately well-funded group could put together the capacity FROM SCRATCH with a moderate investment ($500 K and 3 -6 Ph. Ds)

Group “BW Hacking” Ø Technology access is easy • Robust, world-wide market for used equipment • Simple hardware for all aspects of the technology- could be built from scratch by a few engineers • Chemistry is feasible for companies or laboratories in many (nearly all? ) countries • Molecular biology and bacteriology kits available from many different companies in many countries • Protocols on the internet Ø But, it is still far harder than organism- or tissue culture-based BW hacking • ~$1 M, 3 -6 key technologists, and a modest industrial infrastructure required for synthetic biology

Governance: Select Agent Regulations Ø Screen all orders against a database of select agent genes • Black Watch, Craic Computing Ø Review sequences that are similar to those genes • A Ph. D. reviews several positive hits per day • Most hits are not select agent genes Ø Detailed analysis of select agent genes • • • Check the literature Discuss with customer Decide if we can build the sequence

Current Regulations Require Interpretation Ø Many genes from select agents are not dangerous and are not controlled • E. g. , bacterial metabolic genes Ø Many select agent genes resemble harmless genes • E. g. , non-pathogenic relatives Ø Many scientists use non-functional parts of select agent genes in their research • Viral coat proteins for vaccine development • Enzymes for testing anti-microbial and anti-viral drugs • DNA fragments or proteins for development of diagnostics

Regulatory Clarity is Needed Ø Goals • Restrain/monitor access to dangerous DNA fragments • Retain ability to carry out rapid biomedical and other life science R&D Ø However, no national regulatory scheme can completely block the arrival of new pathogens Ø Moreover, poorly conceived regulation could impede ability to respond to new pathogens

Our Perspective on Regulations Ø Regulation should define the DNA sequences that are covered • Current select agent rules require interpretation Ø And action to be taken when regulated sequences are requested • What needs to be reported? To whom? What is the involvement of our customer in the process? Ø Regulations could shift the development our industry • If regulations require disclosure of all sequence orders, pharmaceutical researchers will not outsource gene synthesis because sequence data is confidential • Such regulation would lead to an instrument (“gene synthesis in a box”) market • The development and dispersion of such instruments would make the technology harder to control

Solution: Select DNA Sequence Database Ø A list of Select DNA Sequences • DNA sequences that could be used to build pathogens or to enhance pathogenicity Ø Actively maintained by an oversight panel and a set of organism-specific experts • Updated on a regular basis (e. g. , monthly) Ø Select sequences defined in terms of a reference sequence and a percentage identity to the reference sequence • Current method of BLAST search against Black. Watch database results in many false positive “hits”, each requires time to research and identify risk

Select Sequences Ø Three classes of sequences • • • Select Agent Genes Related Genes All other genes Require a permit Require reporting No reporting required Ø Control of high-threat sequence Ø Tracking of sequences that could be incorporated into new pathogens • • • Fragments of select agent genes Other pathogenic genes Other sequences? Ø No reporting requirement for most sequences

Operational Considerations Ø Positive requirement to check orders against the Select Sequence database • Current rules make it illegal to provide certain sequences but do not require providers to check for those sequences Ø Clear procedures for identifying organizations and individuals that are authorized to possess molecules encoding Select Sequences Ø Centralized database to collate information on reportable sequences • One could now buy parts of a virus from several different providers and not violate any regulations until they were assembled

Gene Synthesis is an International Industry Ø Researchers are located all over the world Ø Gene synthesis companies exist all over the world • • Dozen + in US Dozen in Europe Several in Asia (at least) Ad hoc (non-commercial) gene synthesis occurs regularly in labs all over the world Ø US regulations cannot block nefarious access to this technology • US regulations can impact the efficiency of our response to pathogens

Rapid, Effective R&D is the Solution Ø Our response to new pathogens depends on decades of basic research AND the immediate application of today’s best technology • Gene synthesis could play an important role in rapid responses to new diseases Ø Scientists working for the good of society have an extremely large advantage in resources • We need to maintain and improve our R&D capacity to respond the this threat Ø Modest investments in current technology could reduce the danger

Summary Ø Gene synthesis and molecular biology are central to modern biological research Ø The technology is ubiquitous and international, thus control from within the USA is not possible Ø Current regulations need improvement • Clear definition of Select Sequences • Tracking of related sequences Ø Poor regulatory choices today could significantly reduce our ability to respond to new pandemics, whether natural or man-made