blue heron biotechnology

Berkley Synthetic Biology Class, April 5, 2006Berkley Synthetic Biology Class, April 5, 2006

Commercial Gene Synthesis TechnologyCommercial Gene Synthesis Technology

John MulliganJohn Mulligan

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TopicsTopics

Commercial gene synthesis todayCommercial gene synthesis today Issues and technology for the futureIssues and technology for the future Governance and the potential for nefarious Governance and the potential for nefarious

applications of synthetic biologyapplications of synthetic biology

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Access to DNA is Central to Modern BiologyAccess to DNA is Central to Modern Biology

Biomedical ResearchBiomedical Research BiologyBiology AgricultureAgriculture New areas such as Synthetic BiologyNew areas such as Synthetic Biology

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Acquiring and Modifying DNA is CostlyAcquiring and Modifying DNA is Costly

Researchers spend > $800MM/year on reagents to Researchers spend > $800MM/year on reagents to clone and modify genesclone and modify genes• Deutsche Banc Alex Brown, 2000Deutsche Banc Alex Brown, 2000

Every $1 spent on reagents represents an Every $1 spent on reagents represents an additional $2 to $5 of fully loaded costsadditional $2 to $5 of fully loaded costs• Labor, overhead, facilities, etc.Labor, overhead, facilities, etc.

Billions of dollars in time and effort every yearBillions of dollars in time and effort every year• Roughly $1 billion in direct costs to NIHRoughly $1 billion in direct costs to NIH• $1-$2 billion to industry$1-$2 billion to industry

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Gene Synthesis Improves Research ProductivityGene Synthesis Improves Research Productivity

Less costly than other methods for many projects Less costly than other methods for many projects today ($1.25 to $1.60 per base pair today)today ($1.25 to $1.60 per base pair today)• Industrial groups believe their internal costs with other Industrial groups believe their internal costs with other

methods to be ~$2 per bpmethods to be ~$2 per bp• Academic groups have lower costs but still find synthesis Academic groups have lower costs but still find synthesis

economical for many projectseconomical for many projects• Cost of synthesis continues to decline rapidlyCost of synthesis continues to decline rapidly

Complete control of sequence allows improved Complete control of sequence allows improved experimental design and new experimental experimental design and new experimental approachesapproaches• Use the perfect gene for your experiment instead of the Use the perfect gene for your experiment instead of the

gene you have in the freezergene you have in the freezer

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Commercial Gene SynthesisCommercial Gene Synthesis

Potentially a substitute for $1 to $2 billion in fully Potentially a substitute for $1 to $2 billion in fully loaded costsloaded costs

We estimate the current market is $20 million to We estimate the current market is $20 million to $30 million a year$30 million a year• Revenues rowing at 30% to 50% a yearRevenues rowing at 30% to 50% a year

- Volume growth much higherVolume growth much higher

• Highly fragmented: 50 or more companies in this area world Highly fragmented: 50 or more companies in this area world widewide

Still a tiny fraction of the overall molecular Still a tiny fraction of the overall molecular biology marketbiology market• We expect it to grow rapidly but to take 5-10 years to reach We expect it to grow rapidly but to take 5-10 years to reach

a significant fraction of the molecular biology marketa significant fraction of the molecular biology market

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Blue Heron Order MixBlue Heron Order Mix

Standard Orders Standard Orders • $500 to $50,000$500 to $50,000• One to ~50 genes, as fast as possibleOne to ~50 genes, as fast as possible• Standard delivery scheduleStandard delivery schedule

High-Volume, Time-Sensitive (Corporate)High-Volume, Time-Sensitive (Corporate)• Hundreds of kilobases, as fast as possibleHundreds of kilobases, as fast as possible• Negotiated delivery scheduleNegotiated delivery schedule• A 200 kb project in 2004 and a 450 kb project in 2005A 200 kb project in 2004 and a 450 kb project in 2005

High-Volume, Time-Insensitive (Government)High-Volume, Time-Insensitive (Government)• Thousands of kilobasesThousands of kilobases• Extended Delivery, discounted priceExtended Delivery, discounted price• Enables full capacity utilization to leverage fixed costs and Enables full capacity utilization to leverage fixed costs and

maximize economies of scalemaximize economies of scale

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Gene Synthesis TechnologyGene Synthesis Technology

In use since the late ’70’s but only beginning to In use since the late ’70’s but only beginning to be widely usedbe widely used

ChallengesChallenges• Error rate: ~1/300Error rate: ~1/300• Mismatched hybridization can lead to scrambled orderMismatched hybridization can lead to scrambled order• Reliability impacts speed and costReliability impacts speed and cost

Three general approachesThree general approaches• ““One pot” ligation and/or PCROne pot” ligation and/or PCR

• Convergent assemblyConvergent assembly

• Solid phase assemblySolid phase assembly

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PCR AssemblyPCR Assembly

Multiple oligonucleotides in a single reaction.

PCR AmplificationPCR Amplification

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PCR AssemblyPCR Assembly

Simple to do, often worksSimple to do, often works• The technology used by nearly all commercial providersThe technology used by nearly all commercial providers• Many published protocolsMany published protocols

LimitationsLimitations• Some sequences are difficult or impossible to PCRSome sequences are difficult or impossible to PCR• Difficult sequences can add to the cost and delivery timeDifficult sequences can add to the cost and delivery time

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Convergent AssemblyConvergent Assembly

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

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Convergent AssemblyConvergent Assembly

A series of simple, reliable reactionsA series of simple, reliable reactions Works on almost any sequenceWorks on almost any sequence But, it is slow and more expensive than PCR-But, it is slow and more expensive than PCR-

based methods for many genesbased methods for many genes

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Inside columnInside column

Solid-Phase AssemblySolid-Phase Assembly

Within each column, double-stranded oligos Within each column, double-stranded oligos (duplexes) are sequentially added to a solid support, (duplexes) are sequentially added to a solid support,

with intervening wash steps.with intervening wash steps.

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Attach duplex to solid phase supportAttach duplex to solid phase support

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WashWash

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Add the second duplex into columnAdd the second duplex into column

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Attach the second duplex to the first duplexAttach the second duplex to the first duplex

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WashWash

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Add the third duplex into columnAdd the third duplex into column

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Attach the third duplexAttach the third duplex

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Repeat to assemble complete fragments Repeat to assemble complete fragments and elute from columnand elute from column

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Simple reaction: two fragments, three endsSimple reaction: two fragments, three ends Drive reaction with molar excessDrive reaction with molar excess Wash away side reactionsWash away side reactions Fully automated at Blue HeronFully automated at Blue Heron

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Solid Phase Assembly of Whole GenesSolid Phase Assembly of Whole Genes

Final Clone

ClonedSequence verifiedRestricted

Subtarget

OligosOligos

SubtargetsSubtargets

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GeneMakerGeneMaker®® Overview Overview

Proprietary software designs Proprietary software designs build strategybuild strategy

Oracle database instructs Oracle database instructs instruments to buildinstruments to build

Oligos are synthesized and Oligos are synthesized and hybridizedhybridized

Patent-pending automated Patent-pending automated solid phase assemblysolid phase assembly

Cloning and sequencing Cloning and sequencing Error removal methods Error removal methods

throughout processthroughout process

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Issues and Technology for the Future Issues and Technology for the Future

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Gene Synthesis is ComplexGene Synthesis is Complex

Every order is differentEvery order is different• Every gene is made from a dozen to several thousand partsEvery gene is made from a dozen to several thousand parts

Every part is new and used for only one orderEvery part is new and used for only one order The smallest parts are chemicalsThe smallest parts are chemicals

• Mixed populations of good and bad partsMixed populations of good and bad parts• Error rate of on in a few hundredError rate of on in a few hundred

Larger parts are biologicalLarger parts are biological• Unpredictable behaviorUnpredictable behavior

The final product must be perfectThe final product must be perfect

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Existing Manufacturing Tools are InadequateExisting Manufacturing Tools are Inadequate

Commodity marketCommodity market• Prices drop 30% to 50% / yearPrices drop 30% to 50% / year• We must drop production costs at least this fastWe must drop production costs at least this fast

Mass customization used in some industriesMass customization used in some industries• Have not found one where every part is newHave not found one where every part is new

Handling high failure rates is critical to Handling high failure rates is critical to controlling manufacturing costscontrolling manufacturing costs

Existing tools focused on assembly-line Existing tools focused on assembly-line production, “job shops”, custom engineeringproduction, “job shops”, custom engineering• None None

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Automated Laboratory vs. ManufacturingAutomated Laboratory vs. Manufacturing

Most or all gene synthesis today is carried out in Most or all gene synthesis today is carried out in sophisticated laboratories with some automationsophisticated laboratories with some automation• PhDs involvedPhDs involved• Difficult to scale rapidlyDifficult to scale rapidly

Within a few years, nearly all commercial gene Within a few years, nearly all commercial gene synthesis will be carried out in manufacturing synthesis will be carried out in manufacturing facilitiesfacilities• Largely automatedLargely automated• Robots for productionRobots for production• People for process developmentPeople for process development• Highly sophisticated process control and schedulingHighly sophisticated process control and scheduling

Interesting, meaty problems for operations Interesting, meaty problems for operations research…research…

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Blue Heron is a Software CompanyBlue Heron is a Software Company

Integrated manufacturing systemIntegrated manufacturing system• Automated storageAutomated storage• Integrated materials handling: e.g., robot arm on a railIntegrated materials handling: e.g., robot arm on a rail• Off the shelf components: pipettors and incubatorsOff the shelf components: pipettors and incubators• Proprietary processProprietary process

Lots of softwareLots of software• Automated design of manufacturing processAutomated design of manufacturing process• Database control to track every fragment and manage Database control to track every fragment and manage

rework cyclesrework cycles• Sophisticated schedulingSophisticated scheduling• Integration software Integration software • Protocol software on individual instrumentsProtocol software on individual instruments

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Nefarious Applications and GovernanceNefarious Applications and Governance

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The Potential for Biowarfare ApplicationsThe Potential for Biowarfare Applications

Many researchers synthesize or clone pathogenic Many researchers synthesize or clone pathogenic DNA as they work to understand the basic DNA as they work to understand the basic biology of the pathogen and to develop new biology of the pathogen and to develop new therapeuticstherapeutics

Most viral genomes are within the range of Most viral genomes are within the range of today’s technologytoday’s technology• Blue Heron delivered the fragments for a >25 kb virus in Blue Heron delivered the fragments for a >25 kb virus in

20042004• Vaccinia is 180 kbVaccinia is 180 kb

- ould be done in 6-12 months, 40 SNPsould be done in 6-12 months, 40 SNPs

One or more bacterial genomes will be One or more bacterial genomes will be synthesized within the next yearsynthesized within the next year

Nefarious uses of synthesis are possibleNefarious uses of synthesis are possible

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Gene Synthesis Technology is WidespreadGene Synthesis Technology is Widespread

““The capacity of this facility is to produce The capacity of this facility is to produce 7.2 tons of phosphoramidite per year… 7.2 tons of phosphoramidite per year…

Currently we have (the) capacity of producing Currently we have (the) capacity of producing 20,000 oligos per day…20,000 oligos per day…

Bioneer offers a special gene synthesis service.”Bioneer offers a special gene synthesis service.”

Bioneer CorporationBioneer Corporation49-3, Munpyeong-dong,49-3, Munpyeong-dong,Daedeok-gu, Daejeon 306-220,Daedeok-gu, Daejeon 306-220,KoreaKorea

But the vast majority of the sophisticated molecularBut the vast majority of the sophisticated molecularbiology capacity is in Europe and North Americabiology capacity is in Europe and North America

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Controlling Synthesis Technology is DifficultControlling Synthesis Technology is Difficult

Synthesis materials are easy to acquireSynthesis materials are easy to acquire• Any sophisticated chemistry group could build Any sophisticated chemistry group could build

oligonucleotide synthesis capacity from scratcholigonucleotide synthesis capacity from scratch• For large-scale synthesis groups the “drop at the bottom of a For large-scale synthesis groups the “drop at the bottom of a

reagent bottle” can add up to kilograms of phosphoramidite reagent bottle” can add up to kilograms of phosphoramidite per year- tracking the materials is not feasibleper year- tracking the materials is not feasible

PCR-based synthesis works on many sequencesPCR-based synthesis works on many sequences Transforming and growing bacteria is low-techTransforming and growing bacteria is low-tech

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New Methods Extend Synthesis CapabilitiesNew Methods Extend Synthesis Capabilities

Build genes with a modified ink-jet printer?Build genes with a modified ink-jet printer?

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Garage Technology in Five Years?Garage Technology in Five Years?

Lone hackers with few resourcesLone hackers with few resources NONO

Governments or organizations Governments or organizations YESYES• Any country or moderately well-funded group could put Any country or moderately well-funded group could put

together the capacity FROM SCRATCH with a moderate together the capacity FROM SCRATCH with a moderate investment ($500K and 3-6 PhDs)investment ($500K and 3-6 PhDs)

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Group “BW Hacking”Group “BW Hacking”

Technology access is easy Technology access is easy • Robust, world-wide market for used equipmentRobust, world-wide market for used equipment• Simple hardware for all aspects of the technology- could be Simple hardware for all aspects of the technology- could be

built from scratch by a few engineersbuilt from scratch by a few engineers• Chemistry is feasible for companies or laboratories in many Chemistry is feasible for companies or laboratories in many

(nearly all?) countries(nearly all?) countries• Molecular biology and bacteriology kits available from many Molecular biology and bacteriology kits available from many

different companies in many countriesdifferent companies in many countries• Protocols on the internetProtocols on the internet

But, it is still far harder than organism- or tissue But, it is still far harder than organism- or tissue culture-based BW hackingculture-based BW hacking• ~$1M, 3-6 key technologists, and a modest industrial ~$1M, 3-6 key technologists, and a modest industrial

infrastructure required for synthetic biologyinfrastructure required for synthetic biology

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Governance: Select Agent RegulationsGovernance: Select Agent Regulations

Screen all orders against a database of select Screen all orders against a database of select agent genesagent genes• Black Watch, Craic ComputingBlack Watch, Craic Computing

Review sequences that are similar to those genesReview sequences that are similar to those genes• A Ph.D. reviews several positive hits per dayA Ph.D. reviews several positive hits per day• Most hits are not select agent genesMost hits are not select agent genes

Detailed analysis of select agent genesDetailed analysis of select agent genes• Check the literatureCheck the literature

• Discuss with customerDiscuss with customer

• Decide if we can build the sequenceDecide if we can build the sequence

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Current Regulations Require InterpretationCurrent Regulations Require Interpretation

Many genes from select agents are not Many genes from select agents are not dangerous and are not controlleddangerous and are not controlled• E.g., bacterial metabolic genesE.g., bacterial metabolic genes

Many select agent genes resemble harmless Many select agent genes resemble harmless genesgenes• E.g., non-pathogenic relativesE.g., non-pathogenic relatives

Many scientists use non-functional parts of select Many scientists use non-functional parts of select agent genes in their researchagent genes in their research• Viral coat proteins for vaccine developmentViral coat proteins for vaccine development• Enzymes for testing anti-microbial and anti-viral drugsEnzymes for testing anti-microbial and anti-viral drugs• DNA fragments or proteins for development of diagnosticsDNA fragments or proteins for development of diagnostics

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Regulatory Clarity is Needed Regulatory Clarity is Needed

GoalsGoals• Restrain/monitor access to dangerous DNA fragmentsRestrain/monitor access to dangerous DNA fragments• Retain ability to carry out rapid biomedical and other life Retain ability to carry out rapid biomedical and other life

science R&Dscience R&D

However, no national regulatory scheme can However, no national regulatory scheme can completely block the arrival of new pathogenscompletely block the arrival of new pathogens

Moreover, poorly conceived regulation could Moreover, poorly conceived regulation could impede ability to respond to new pathogensimpede ability to respond to new pathogens

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Our Perspective on RegulationsOur Perspective on Regulations

Regulation should define the DNA sequences that are Regulation should define the DNA sequences that are coveredcovered• Current select agent rules require interpretationCurrent select agent rules require interpretation

And action to be taken when regulated sequences are And action to be taken when regulated sequences are requestedrequested• What needs to be reported? To whom? What is the involvement of What needs to be reported? To whom? What is the involvement of

our customer in the process?our customer in the process?

Regulations could shift the development our industryRegulations could shift the development our industry• If regulations require disclosure of all sequence orders, If regulations require disclosure of all sequence orders,

pharmaceutical researchers will not outsource gene synthesis pharmaceutical researchers will not outsource gene synthesis because sequence data is confidentialbecause sequence data is confidential

• Such regulation would lead to an instrument (“gene synthesis in a Such regulation would lead to an instrument (“gene synthesis in a box”) market box”) market

• The development and dispersion of such instruments would make The development and dispersion of such instruments would make the technology harder to controlthe technology harder to control

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Solution: Select DNA Sequence DatabaseSolution: Select DNA Sequence Database

A list of Select DNA Sequences A list of Select DNA Sequences • DNA sequences that could be used to build pathogens or to DNA sequences that could be used to build pathogens or to

enhance pathogenicityenhance pathogenicity

Actively maintained by an oversight panel and a Actively maintained by an oversight panel and a set of organism-specific expertsset of organism-specific experts• Updated on a regular basis (e.g., monthly)Updated on a regular basis (e.g., monthly)

Select sequences defined in terms of a reference Select sequences defined in terms of a reference sequence and a percentage identity to the sequence and a percentage identity to the reference sequencereference sequence• Current method of BLAST search against BlackWatch Current method of BLAST search against BlackWatch

database results in many false positive “hits”, each requires database results in many false positive “hits”, each requires time to research and identify risktime to research and identify risk

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Select SequencesSelect Sequences

Three classes of sequencesThree classes of sequences• Select Agent GenesSelect Agent Genes Require a permit Require a permit • Related GenesRelated Genes Require reportingRequire reporting• All other genesAll other genes No reporting requiredNo reporting required

Control of high-threat sequenceControl of high-threat sequence Tracking of sequences that could be incorporated Tracking of sequences that could be incorporated

into new pathogensinto new pathogens• Fragments of select agent genesFragments of select agent genes

• Other pathogenic genesOther pathogenic genes

• Other sequences?Other sequences?

No reporting requirement for most sequencesNo reporting requirement for most sequences

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Operational ConsiderationsOperational Considerations

Positive requirement to check orders against the Positive requirement to check orders against the Select Sequence databaseSelect Sequence database• Current rules make it illegal to provide certain sequences but Current rules make it illegal to provide certain sequences but

do not require providers to check for those sequencesdo not require providers to check for those sequences

Clear procedures for identifying organizations Clear procedures for identifying organizations and individuals that are authorized to possess and individuals that are authorized to possess molecules encoding Select Sequencesmolecules encoding Select Sequences

Centralized database to collate information on Centralized database to collate information on reportable sequences reportable sequences • One could now buy parts of a virus from several different One could now buy parts of a virus from several different

providers and not violate any regulations until they were providers and not violate any regulations until they were assembledassembled

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Gene Synthesis is an International IndustryGene Synthesis is an International Industry

Researchers are located all over the worldResearchers are located all over the world Gene synthesis companies exist all over the Gene synthesis companies exist all over the

worldworld• Dozen + in USDozen + in US• Dozen in EuropeDozen in Europe• Several in Asia (at least)Several in Asia (at least)• Ad hoc (non-commercial) gene synthesis occurs regularly in Ad hoc (non-commercial) gene synthesis occurs regularly in

labs all over the worldlabs all over the world

US regulations cannot block nefarious access to US regulations cannot block nefarious access to this technologythis technology• US regulations can impact the efficiency of our response to US regulations can impact the efficiency of our response to

pathogenspathogens

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Rapid, Effective R&D is the SolutionRapid, Effective R&D is the Solution

Our response to new pathogens depends on Our response to new pathogens depends on decades of basic research AND the immediate decades of basic research AND the immediate application of today’s best technologyapplication of today’s best technology• Gene synthesis could play an important role in rapid Gene synthesis could play an important role in rapid

responses to new diseasesresponses to new diseases

Scientists working for the good of society have Scientists working for the good of society have an extremely large advantage in resourcesan extremely large advantage in resources• We need to maintain and improve our R&D capacity to We need to maintain and improve our R&D capacity to

respond the this threatrespond the this threat

Modest investments in current technology could Modest investments in current technology could reduce the dangerreduce the danger

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SummarySummary

Gene synthesis and molecular biology are central Gene synthesis and molecular biology are central to modern biological researchto modern biological research

The technology is ubiquitous and international, The technology is ubiquitous and international, thus control from within the USA is not possiblethus control from within the USA is not possible

Current regulations need improvementCurrent regulations need improvement• Clear definition of Select SequencesClear definition of Select Sequences• Tracking of related sequencesTracking of related sequences

Poor regulatory choices today could significantly Poor regulatory choices today could significantly reduce our ability to respond to new pandemics, reduce our ability to respond to new pandemics, whether natural or man-madewhether natural or man-made