Berkley Synthetic Biology Class, April 5, 2006

1
Berkley Synthetic Biology Class, April 5, 2006
Commercial Gene Synthesis Technology John
Mulligan
2
Topics

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

3
Access to DNA is Central to Modern Biology

• Biomedical Research
• Biology
• Agriculture
• New areas such as Synthetic Biology

4
Acquiring and Modifying DNA is Costly

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

5
GeneMaker Gene Synthesis Can Provide Any
Sequence

• Customer orders via secure website
• Blue Heron manufactures and ships DNA molecule(s)
• About 2 to 4 weeks later, customer receives
  exactly the DNA they wanted
• Customers use genes in biomedical RD

almost
longer for big genes
6
Gene Synthesis Improves Research Productivity

• 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
• 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

7
Commercial Gene Synthesis

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

8
Blue Heron Order Mix

• Standard Orders
• 500 to 50,000
• One to 50 genes, as fast as possible
• Standard delivery schedule
• 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
• 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

9
Gene Synthesis Technology

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

10
PCR Assembly
Multiple oligonucleotides in a single reaction.
11
PCR Assembly

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

12
Convergent Assembly
A series ligation and purification steps, each
involving only two fragments.
13
Convergent Assembly

• A series of simple, reliable reactions
• Works on almost any sequence
• But, it is slow and more expensive than PCR-based
  methods for many genes

14
Solid-Phase Assembly
Within each column, double-stranded oligos
(duplexes) are sequentially added to a solid
support, with intervening wash steps.
15
Solid-Phase Assembly
Attach duplex to solid phase support
16
Solid-Phase Assembly
Wash
17
Solid-Phase Assembly
Add the second duplex into column
18
Solid-Phase Assembly
Attach the second duplex to the first duplex
19
Solid-Phase Assembly
Wash
20
Solid-Phase Assembly
Add the third duplex into column
21
Solid-Phase Assembly
Attach the third duplex
Inside column
22
Solid-Phase Assembly
Repeat to assemble complete fragments and elute
from column
23
Solid-Phase Assembly

• Simple reaction two fragments, three ends
• Drive reaction with molar excess
• Wash away side reactions
• Fully automated at Blue Heron

24
Solid Phase Assembly of Whole Genes
25
GeneMaker Overview

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

26
Applications of Gene Synthesis

• Three years ago
• Hard to clone cDNAs
• Codon optimization
• Designed proteins
• In 2004
• Outsource some or all cloning
• Large genes that do not PCR well (gt2kb)
• Large constructs (gt25 kb)
• Vectors
• Future
• Synthetic biology, engineered genomes, ??
• Half or more of the consumption of gene synthesis
  in 10 years will be for applications that we are
  not thinking about today

27
Issues and Technology for the Future
28
Gene Synthesis is Complex

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

29
Existing Manufacturing Tools are Inadequate

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

30
Automated Laboratory vs. Manufacturing

• Most or all gene synthesis today is carried out
  in sophisticated laboratories with some
  automation
• PhDs involved
• Difficult to scale rapidly
• 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
• Interesting, meaty problems for operations
  research

31
Blue Heron is a Software Company

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

32
Nefarious Applications and Governance
33
The Potential for Biowarfare Applications

• Many researchers synthesize or clone pathogenic
  DNA as they work to understand the basic biology
  of the pathogen and to develop new therapeutics
• Most viral genomes are within the range of
  todays technology
• Blue Heron delivered the fragments for a gt25 kb
  virus in 2004
• Vaccinia is 180 kb
• ould be done in 6-12 months, 40 SNPs
• One or more bacterial genomes will be synthesized
  within the next year
• Nefarious uses of synthesis are possible

34
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
35
Controlling Synthesis Technology is Difficult

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

36
New Methods Extend Synthesis Capabilities
Build genes with a modified ink-jet printer?
37
Garage Technology in Five Years?

• Lone hackers with few resources NO
• Governments or organizations YES
• Any country or moderately well-funded group could
  put together the capacity FROM SCRATCH with a
  moderate investment (500K and 3-6 PhDs)

38
Group BW Hacking

• 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
• But, it is still far harder than organism- or
  tissue culture-based BW hacking
• 1M, 3-6 key technologists, and a modest
  industrial infrastructure required for synthetic
  biology

39
Governance Select Agent Regulations

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

40
Current Regulations Require Interpretation

• Many genes from select agents are not dangerous
  and are not controlled
• E.g., bacterial metabolic genes
• Many select agent genes resemble harmless genes
• E.g., non-pathogenic relatives
• 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

41
Regulatory Clarity is Needed

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

42
Our Perspective on Regulations

• Regulation should define the DNA sequences that
  are covered
• Current select agent rules require interpretation
• 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?
• 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

43
Solution Select DNA Sequence Database

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

44
Select Sequences

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

45
Operational Considerations

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

46
Gene Synthesis is an International Industry

• Researchers are located all over the world
• 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
• US regulations cannot block nefarious access to
  this technology
• US regulations can impact the efficiency of our
  response to pathogens

47
Rapid, Effective RD is the Solution

• Our response to new pathogens depends on decades
  of basic research AND the immediate application
  of todays best technology
• Gene synthesis could play an important role in
  rapid responses to new diseases
• Scientists working for the good of society have
  an extremely large advantage in resources
• We need to maintain and improve our RD capacity
  to respond the this threat
• Modest investments in current technology could
  reduce the danger

48
Summary

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