Biotechnology

1

Lecture 12 misc and ELSI

• Policy, public safety, research and Frankenfood
• Ag and biotech updates
• New drugs from biodiversity/
• Ethics and politics, and religion
• Antibiotics, biotechnology and molecular
  biotechnology
• Patents/Ethics
• de novo life/Synthetic Genomics

2

Asilomar Conference on Recombinant DNA

• 1972 Berg Lab experiment designed to clone SV40
  fragments into lambda
• Then to introduce into E. coli, which is a human
  pathogen (this part not completed)
• one concern is that escaped recombinant might
  generate cancer in hosts
• Concerns about potential biohazards, a group of
  researchers sent letter to NAS
• Convened committee to review in 1974
• Recommended an international conference and to
  halt on-going expts

• wikipedia

• Photo http//www.californiacoastline.org/cgi-bin/
  image.cgi?image1081modesequentialflags0

3

Asilomar Conference on Recombinant DNA

• Feb 75 organized by Paul Berg
• 140 professionals including (mostly) biologists,
  lawyers and physicians
• Discuss potential biohazards and regulation of
  biotechnology
• Draw up voluntary guidelines to ensure the
  safety of recombinant DNA technology
• Prior, due to potential safety hazards,
  scientists worldwide had halted experiments
• using recombinant DNA technology

• wikipedia

• Photo http//www.visitasilomar.com/

4

Asilomar Conference on Recombinant DNA
Principles guiding how to conduct recombinant DNA
technology expts 1. Containment should be made
an essential consideration in the exptl design 2.
Effectiveness of the containment should match
the estimated risk as closely as
possible Suggested use of biological barriers to
limit the spread of recombinant DNA Vectors that
were able to grow in only specified
hosts Physical containment hoods, limited access
or negative pressure labs Good microbiological
practices to limit organism escape Education and
training of all personnel

• wikipedia

• Photo http//nobelprize.org/nobel_prizes/chemist
  ry/articles/berg/index.html

5

Immediate sanctions

• Strange case of the virus that was miscloned
• Sci Apr 3, 1981 page 27
• UCSD. IKennedy. Cloned Semliki Forest virus
• Class 3 agent (smallpox class), depending on
  conditions of use and
• geographical location of the laboratory
• Usually produces only a mild fever in man and has
  caused only one death
• Done Jan (or March/April) 1980
• Trying to beat the competition before the start
  date?
• PI first said it was a case of sabotage, then
  considers cross-contamination when
• shipped from Univ of Warwick, from where he
  moved
• Was repeating the fingerprint when his students
  felt the Chairman should be notified
• a procedure then but not now 1981 in
  violation of the NIHs
• recombinant DNA guidelines.
• The cloning violation was perhaps of no great
• significance compared with the fact that
  Kennedy had,
• for whatever reason, come to lose the trust
  of his colleagues.
• No record of MOU filed with IBC

6

Frankenfood

• Mar-Apr2000 Mother Jones (Bill McKibben).
  Muggles in the Ozone
• RE Last winters protest in Seattle
• .it was the most significant protest in America
• since the waning days of Vietnam
• Sometimes it seemed as if no two protestors were
  holding the same sign

• Photo http//www.cpinternet.com/mbayly/facesofre
  sistance2.htm
• http//www.motherjones.com/commentary/power_plays/
  2000/03/mckibben.html

7

Agriculture and molecular biotech update

• Proposed ban on genetically modified corn in
  Europe
• Nov 23, 2007
• EU environmental officials have determined that
  two kinds of GM corn could harm butterflies
• Affect food chains and disturb life in rivers and
  streams
• Environment commissioner contends GM corn could
  affect certain butterfly species
• larvae of the monarch behave differently than
  other larvae.
• Ban on sale seeds made by DuPont-Pioneer, Dow
  Agrosciences and Syngenta
• Aug07 protest in France against GM crops
• Modified corn grown in US for years
• 2005 European Food Safety Authority, based in
  Parma
• Ruled the products were unlikely to harm human
  and animal health or the environment
• Crops grown using GM corn already imported into
  several European countries, including
• France and Germany, and used to feed cows and
  chickens

8

Agriculture and molecular biotech update

• Reuters, 10/25/07
• European Union authorized imports of four GM
  crops
• 27 national markets for next 10 years
• Three are corn, two hybrids, and one is sugar
  beet
• None to be grown in Europe
• Imported as food and animal feed
• Corn and one hybrid by Pioneer/Hi-Bred Intl
  (DuPont)
• Others by Monsanto, and Monsanto and German KWS
  SAAT

9

Biotech foods, processed

• LATimes, 10/22/07 Biotech foods are still hard
  to swallow
• More than 70 of processed foods such as flour,
  cereal, chips
• and cookies contain biotech ingredients
• Second-generation
• Hypoallergenic peanuts, vitamin-rich rice,
  folate-rich tomatoes, calcium-filled potatoes
• Originally, goals were
• Crops that did not rot, spoil, die from frost
• Boost harvests, feed the hungry and fortify the
  malnourished
• Frankenfoods man-made aberrations
• But, mostly found in processed foods via corn,
  soy and canola
• Used to withstand herbicides and to resist pests
• Exception virus-resistant papaya from Hawaii

10

Biotech foods

• LATimes, 10/22/07 Biotech foods are still hard
  to swallow
• GM vs conventional breeding
• 1991 cold-tolerance gene from flounder into
  tomato for frost-resistant failed
• But 60 US corn contain Bt gene, against European
  corn borer
• 90 soy has genes from other bacteria for
  herbicide resistance
• Lemaux, UCB, sorghum- African crop, increase
  amino acids, vitamin and mineral content
• 2000 daffodil genes into rice, with 23x beta
  carotene, vitA
• 1990s FDA new allergens created
• 1996 Brazil nut gene into soybean to be more
  nutritious but triggered nut allergies
• 2000 Starlink corn contained protein that may be
  an allergen, made its way into food chain -taco
  shell
• Allergies when heard about it, but no link proven

11

Finding new drugs, antibiotics, (anti-cancer
cells)

• It's a simple proposition A medicine is merely
  a compound that repels or kills or somehow
  interferes
• with the organisms and processes of disease.
  
• In short, it is chemistry. Medicines from
  aspirin to penicillin are natural chemicals
  harnessed
• for the benefit of countless millions of
  humans.
• (LATimes 5/18/06. JBalzar Neptunes Medicine
  Chest)

12

Biodiversity Gold in Yellowstones Microbes

• Yellowstone Parks steam vents and Hot Springs
• Unique microbes extremophiles
• Implications in medicine, agriculture and energy
  (basic research), (biotechnology)
• 1966 TBrock gathered samples of gt80C organisms
  from pink algae and microbial mats of Lower
  Geyser Basin
• Thermus aquaticus to ATCC available for 35
• Taq-based technology sold to Hoffman-LaRoche for
  300M
• Annual sales today of licenses and equipment run
  over 200M
• 2006 new species of bacterium that produces
  chlorophyll
• Unique grasses around hot springs, with symbiosis
  with heat-tolerant fungus (drought-resistant
  plants)
• 1998 research-sharing agreement with Diversa
  Corp. bio-prospecting
• Disputed by non-profit groups as bio-piracy
• TBrock Yellowstone didnt get any money from
  it. I didnt get any money, either, and Im not
  complaining.
• The Taq culture was provided for public
  research use, and it has given great benefit to
  mankind.

13

Harvesting from the oceans, biodiversity

• W Fenical, Scripps Institute of Oceanography (Ctr
  for Marine Biotech and Biomed)/University of
  California
• Founded Nereus Pharmaceuticals
• 1983 Carribean sea whip (Bahamas)
• anti-inflammatory and analgesic metabolites,
  pseudopterosin
• cosmetic rights to Estee Lauder
• 94-95, amount UCs top ten royalty earners
  _at_680,000
• First time, a scientifically proven marine
  product
• UCSB as treatment for wounds, countering
  reactions swelling and inflammation to allow
  faster healing
• Due to pharmaceutical potential and cosmetic
  applications, we have been active in the
  development of
• biotechnological production methods of the
  pseudopterosins.
• SalA, anti-cancer drug from deep sea floor
  bacterium testing for blood and bone cancers
• NPI-0058, anti-cancer drug from seaweed fungus
  testing for aggressive tumors in lung, breast and
  pancreas
• 13 other compounds in trials 2001, DNA of
  bacteria from sampling of sea floor- no hits with
  GenBank

14

Finding new drugs Nereus
15

Approving new drugs

• morning after pill designed to prevent
  pregnancy if taken within 72hrs
• approved by science panel for OTC
• approved by two senior FDA officials
• overruled by FDA chief, then acting/then
  permanent (finally, left)
• Lester Crawford
• circumventing normal practices to delay
  indefinitely drug approval for further study
• nonpartisan congressional inquiry Nov05,
• unusual involvement of the commissioners
  office
• Should be based on medical science, eg, data and
  efficacy not politicized
• Similar worry for Guardisil, 2006
• (WAPost 5/25/06)

16

Infectious diseases, epidemics
17

Infectious disease vs vaccines (one approach)
((vs antibiotics))

• Four Horsemen of the Apocalypse

• http//pathmicro.med.sc.edu/lecture/vaccines.htm

18

Infectious diseases which is more
dangerous/scary?

• One is graphic, one is not- at the beginning
• 11/29/07 msnbc New deadly strain of Ebola
  emerges
• 12/3/07 msnbc Outbeak is still ongoing
• Democratic Republic of Congo 51 infected, 16
  dead (31)
• Analysis show it is a previously unknown strain
• Last major Congo outbreak in 1995 killed 245
  people
• 2000 Uganda killed gt170 people
• Ebola virus first emerged in 1976, simultaneous
  outbreaks in Sudan and Zaire
• Zoonotic virus hemorrhagic fever
• Four identified strains, Zaire, Sudan, Reston,
  Cote dIvoire (1 case)
• Zaire strain killed 80 (-90?) Sudan killed 50
• Incubation period of 15 days
• No treatments

• wikipedia
• http//images.google.com/imgres?imgurlhttp//www.
  nlm.nih.gov/medlineplus/ency/images/ency/fullsize/
  17160.jpgimgrefurlhttp//www.nlm.nih.gov/
• medlineplus/ency/imagepages/17160.htmh320w400
  sz18tbnid-DFyXPEyexIBTMtbnh99tbnw124prev
  /images3Fq3Debola26um
• 3D1start1saXoiimagesctimagecd1

19

Complex disease, complex solution

• One is graphic, one is not- at the beginning
• Consumption
• John Henry Doc Holliday. Aug 14, 1851- Nov 8,
  1887
• 1866 mother died of TB
• 1872 started dentistry practice
• Diagnosed with TB (not known to be contagious-
  just fatal),
• given a few months to live
• Moved to drier warmer southwest US
• Changed lifestyle, more violent. but
• Died in bed of TB

• Wikipedia photo http//www.trainweb.org/chris/o
  pt0015.jpg

20

Complex disease, complex solution

• One is graphic, one is not- at the beginning
• TB bacilli doubling time days or weeks, growth
  within cells
• May or may not cause symptoms
• Common to be infected with dormant bacteria and
  never become sick
• Current treatment 40-yr old drug regime
• Combination of 4-6 antibiotics taken over 6-9
  months
• MDR-TB, resistance to at least two of first-line
  anti-TB drugs
• Second-line drugs taken over 1-2 year period
• If treatment disrupted, then could develop
• XDR-TB strains
• First identified in Spring 06, XDR-TB is
  resistant to gt 3 of the six classes
• of second-line drugs leaving XDR-TB virtually
  untreatable
• Diagnosis of TB through sputum smear exam, in use
  for 125 years (not optimal)

• http//www.ngpharma.com/pastissue/article.asp?art
  271778issue225 photo wikipedia

21

Pulmonary tuberculosis re-emerging

• May 29, 2007 US seeks fliers possibly exposed
  to rare TB
• (11/28/07 msnbc none of 250 passengers tests
  positive)
• (11/28/07 msnbc "I feel like I've always felt,"
  Speaker said Tuesday. "I feel fine.)
• Quarantine order was first since US govt
  quarantined patient with smallpox in 1963
• TB spread through air, affecting lungs, killing
  2M people per year
• TB rate in US had fallen to low of 13,767 in
  2006 affecting 4.6 per 100,000 US
• Second generation drugs isoniazid and rifampin
  (-gt multidrug-resistant, MDR)
• (XDR extensively drug-resistant)
• Global concerns in the Jet Age
• 2007 Mexican citizen travels between US and
  Mexico despite TB diagnosis

22

Pulmonary tuberculosis Global concerns

• Oct 18, 2007 WATimes Mexican citizen travels
  between US and Mexico despite TB diagnosis
• Highly contagious form, MDR-TB
• Crossed US border 76x, multiple domestic flights
  in past year
• Customs and Border Protection agency warned 4/16
  Homeland Security six weeks to send alert
• PS, used different names

23

Drug-resistant tuberculosis pandemic

• MDR-TB and XDR cases growing among minority and
  people born outside US
• 2005 CDC tallied 14,097 TB cases in US, with 124
  as MDR TB
• 1993 to 2006, 49 cases in US 0.5M in world as
  XDR
• Treatment for XDR case averages 500,000,
  exceeding 2M
• WHO XDR TB reported in 37 countries
• South Africa KwaZulu-Natal province outbreak
  XDR TB killing 52/53 patients with AIDS in 25d
• Pandemic?
• Globally, 96 of all TB treatable with the four
  drugs in std regime
• (former Soviet Union, some countries showing
  MDR-TB up to 20)
• (Russia, China, India and SAfrica worst-hit with
  MDR/XDR at 60 of worlds cases)
• (reminiscent of pre-antibiotic era of 1943)
• Globally, 4 are MDR

24

Drug-resistant tuberculosis society/ethics

• msnbc 4/2/07 Involuntary detention,
  Quarantine
• Drug-resistant TB raises ethical dilemma
• Phoenix county hospital, jail cell 27-yr old TB
  patient in cell with negative pressure
  ventilation system
• not charged with a crime, but locked up
  indefinitely due to extensively drug-resistant
  XDR-TB
• Virtually untreatable, court ordered lock-up
  because did not heed instructions to wear a mask
  in public
• unfair to be treated this way
• (lived in Russia for 15 years, returned to US
  last year after diagnosis)
• said he realizes now that he endangered the
  public.
• I thought Id come to a country where Id
  finally be treated like a person, and bam, here I
  am.
• US in 2006 had 13,767 reported cases of TB
• 2007 Texas has placed 17 into an involuntary
  quarantine facility
• Some run out of options and need to be
  quarantined for the rest of their lives
• One lived 8 years in SC before dying of TB
  escaping once from home detention
• should detain people if they are uncooperative
  Were on the verge of taking what was a
• Curable disease, one of the best known diseases
  in human endeavors, and making it incurable.
• RUpshur, Joint Centre for Bioethics/ UToronto
• Same ethical dilemma generations ago with leprosy
  and smallpox
• Now, XDR-TB, drug-resistant staph infections,
  pandemic flu

25

Drug-resistant M. tuberculosis comparative
genomics

• 11/20/07 msnbc
• Broad Institute/MIT
• Sequenced genomes of XDR-TB and MDR
  (multidrug-resistant) TB, other strains
• Found a few mutations which may explain
  drug-resistance
• Mycobacterium tuberculosis infects up to 2B
  people
• Most have latent or inactive infections
• 2005 8.8M became infected 1.6M died of it
• Estimated 500k have MDR-TB
• XDR-TB kills 85 of afflicted
• Can take weeks to diagnose standard TB or MDR or
  XDR strains

26
KILLER BUG ATE MY FACE

• flesh-eating bacteria -necrotizing fascitis
• Streptococcus pyogenes
• Destruction of skin and muscles via toxins
  (virulence factors)
• Fast-spreading infections mortality rate 30
  rare public in 1990s
• Treatment includes IV penicillin, vancomycin and
  clindamycin
• Including aggressive debridement, amputation
• Oct06 EMBO Journal. EHanski. Mechanism of
  protein blocking immune system signals
• Mouse model SilCR turned off in M14 virulent
  strain add back to mouse and survivable
• SilCR down-regulates ScpC which destroys host
  IL-8
• 2004 rarer more serious form observed, as a
  strain of Staphylococcus aureus
• Resistant against methicillin
• Super Strep appeared in Ohio and Texas in 92/93
  in 140 people
• 12 hrs to incapacitate most, and caused 3 days of
  high fevers mortality at 10

• Wikipedia photo http//commtechlab.msu.edu/site
  s/dlc-me/news/ns195dis1.html

27

Antibiotic-resistant staph infection

• Oct 07. Reuters. New strain of strep emerges
  as major US infection
• Major cause of childhood infections. But even
  drug-resistant versions can be killed with right
  antibiotics
• Should be aware and switch antibiotics for
  children with severe infections who do not
  respond quickly
• Type of Steptococcus pneumonia, strain 19A,
  causing 40 of pneumococcal infections in
  children
• 15 resistant to ceftriaxone use vancomycin
• Also, increasing numbers of infections with
  drug-resistant superbugs
• JAMA Oct07 methicillin-resistant Staph aureus,
  MRSA, killed 18,650 in 2005 and
• made 94,360
  seriously sick
• Mortality rate exceeds
  HIV/AIDS for 2005
• MRSA outbreak killing one caused 21 VA schools to
  close in Oct
• amednews.com CDC report in recent weeks,
  deaths of preschooler in NH 11-yr old in MS
• 12-yr old in NY and 17-yr old in VA.
  Pittsburgh nine football players in one school
  with MRSA
• There is still a group of doctors who dont
  culture in the interest of costs.
• MRSA is part of the bigger problem, accounting
  for only 10 of health care-associated
  infections
• C. difficile, Acinetobacter (returning from
  Iraq), Klebsiella -gt all with multidrug-resistance
  
• Return to the pre-antibiotic era

28

Antibiotic-resistant staph infection Mouse model

• 10/17/07 WAPost Drug-resistant Staph germs toll
  is higher than thought
• (CDC report/JAMA)
• 11/11/07 msnbc
• Methicillin-resistant Staphylococcus aureus, MRSA
• First-line antibiotics, eg penicillin family
• Previously associated with health care facilities
  (HA-MRSA) and immunocompromised individuals
• Recent cases, aggressive strain,
  community-acquired MRSA or CA-MRSA
• Severe infections and deaths of otherwise healthy
  people
• CA-MRSA secretes a peptide that causes immune
  cells, neutrophils, to burst
• Genes for the peptides found in both MRSA and
  CA-MRSA but expressed more in CA-MRSA
• Mouse and human blood 5 min exposure causes
  neutrophils to flatten and show signs of membrane
• damage. After 60 min, many cells had
  disintegrated completely
• (MOtto, et al. Nature Med Nov07) specifically
  identifying a factor which seemingly makes
• CA-MRSA more pathogenic than HA-MRSA
• Only 14 of MRSA are CA-MRSA, recent months have
  been found in schools, including

29

Antibiotics biotechnology

• Nature May06. Merck Pharma
• Problem Rapid resistance to existing
  antibiotics recent emergence of superbugs
• One potential solution Biodiversity, eg, screen
  soil from South Africa
• Streptomyces platensis
• platensimycin
• Approach test extracts with bacterium with
  genetic defect in metabolic pathway
• not targeted by current
  antibiotics
• 250,000 extracts
• mice infected with problematic strain of
  Staphylococcus aureus

30

Antibiotics biotechnology

• Grace Yim
• Graphics Fan Sozzi

31

Antibiotics biotechnology

• Problem Rapid resistance to existing
  antibiotics recent emergence of superbugs
• Paradigm shift from due to spontaneous random
  mutation to
• Horizontal gene transfer
• Misuse of antibiotics
• Underuse of antibiotics
• Farmyard biotech use of antibiotics
• Natural products and natural selection
• Public health and sewage treatment

• Grace Yim
• Graphics Fan Sozzi

32

Antibiotics biotechnology

• Problem Rapid resistance to existing
  antibiotics recent emergence of superbugs
• 1944 General clinical use of penicillin
• Five years ago (ca. 2000), 150 drugs, with new
  ones every 8-10 years, but
• many hit similar targets

• Grace Yim
• Graphics Fan Sozzi

33

Penicillin Natural Products

• Ancient Greece, India- molds and plants to
  treat infection China- moldy bean curd on cuts
• 1929. AFleming, Penicillium mold must have an
  antibacterial substance
• Isolated and named active substance, penicillin,
  from halo of inhibition of bacterial growth
• around a contaminant blue-green mould on a
  Staphylococcus plate culture.
• Unsuccessful attempts to recruit chemist to
  synthesize for mass production
• HWFlorey et al (1938)/Moyer, Coghill, Raper
  (1941-3)/JKane, Pfizer scientists (1941-4)
• Large quantities of pharmaceutical-grade
  penicillin

• wikipedia

34

Methicillin Synthetic Products

• Organic chem synthesis
• Narrow spectrum beta-lactam antibiotic of the
  penicillin class
• Beecham 1959
• Previously used to treat susceptible
  Gram-positive, particularly S. aureus
• Inhibits cell wall synthesis
• Competitively inhibits transpeptidase,
  cross-links D-Ala-Ala, as a structural analog

• wikipedia

35

Vancomycin Natural Products

• Glycopeptide drug of last resort
• Last-line antibiotic for serious Gram-positive
  infections
• Strong effect on Gram-positive, Streptococci,
  Staphylococci and C. difficile
• Resistant to penicillin and cephalosporin
• Effect on MRSA (S. aureus)
• Resistance will result in return to era of fatal
  bacterial infections
• 1990s-2000s VISA, vancomycin-intermediate S.
  aureus
• VRSA, vancomycin-resistant S. aureus
  vancomycin-resistant C. difficile
• First isolated by ECKornfeld _at_Eli Lilly, from
  soil sample collected from the
• interior jungles of Borneo by a missionary
• Fast-tracked approval FDA in 1958, due to
  penicillin-resistance

• wikipedia

36

Vancomycin Optimization

• H-MJungJ-KLee, et al. 2007. Biotech Prod
  Process Engr. Optimization of culture
  conditions and
• scale-up to pilot and plant scales for
  vancomycin production by Amycolatopsis
  orientalis
• High vancomycin producing strain, previoulsy
  Streptomyces isolated from Borneo soil

37

Vancomycin Optimization

• H-MJungJ-KLee, et al. 2007. Biotech Prod
  Process Engr. Optimization of culture
  conditions and
• scale-up to pilot and plant scales for
  vancomycin production by Amycolatopsis
  orientalis
• From lab scale at 7L to pilot scale 300L to plant
  scale 5,000L

38

Vancomycin Optimization

• H-MJungJ-KLee, et al. 2007. Biotech Prod
  Process Engr. Optimization of culture
  conditions and
• scale-up to pilot and plant scales for
  vancomycin production by Amycolatopsis
  orientalis
• From lab scale at 7L to pilot scale 300L to plant
  scale 5,000L

39

Drugs of last resort antibiotics

• Drugs used only when all other options are
  exhausted
• Antibiotics, antivirals or chemotherapy agents
• Have most potent effects and/or are drugs for
  which no or very few strains are known
• Usually withheld to prevent development of
  resistance or due to unpleasant side effects
• Amikacin aminoglycoside antibiotic that binds
  to 30S ribosomal subunit
• Imipenem IV beta-lactam antibiotic, developed
  1985 Broad spectrum Gorilla-cillin
• Linezolid synthetic antibiotic, first of
  oxazolidinone class inhibits protein synthesis
  initiation
• Vancomycin glycopeptide antibiotic cell wall
  inhibitor

• wikipedia

40
Patents, and enforcement

• A patent is a set of exclusive rights granted by
  a state to an inventor or his assignee
• for a fixed period of time in exchange for a
  disclosure of an invention
• A patent is not a right to practice or use
  invention.
• A patent provides the right to exclude others
  from making, using, selling, offering
• for sale, or importing the patented invention
  for the term of the patent
• Agreement to share the details of the invention
  with the public
• PCR and real-time PCR Filed June 1998
• 3/29/04 MJ Research filed for bankruptcy court
  protection
• 4/19/04 Applied Biosystems and Roche Molecular
  Systems win patent infringement
• litigation against MJ Research, Inc and its
  principals, Michael and John Finney
• Awarded damages of 19.8M plus enhancement of
  damages, including legal fees, since
• several infringements were found to be willful,
  ie doubled
• 4/4/05 MJ Research, a division of Bio-Rad, to pay
  additional 15M in damages, totaling 35M
• 2/13/06 Settled with Bio-Rad Labs

41
Patents based on biotechnology

• LATimes 11/28/07
• Dr. JRCade, Gatorade
• Professor of medicine and physiology research on
  kidney and liver disease,
• diabetes, hypertension and schizophrenia
• 1965 Developed for U of Florida, Gainsville
  football team to stay hydrated
• Inspired multimillion-dollar sports beverage
  industry
• 2006 Gatorade held more than 80 of 7.5B/yr US
  sports drink market
• World-wide sales in the billions
• Since 1973, UF collected gt150M for its 20 share
  of royalties
• From a comment that a player was not urinating
  during football practice
• Loss of 10 pounds of sweat, eg carbohydrates and
  electrolytes
• Collected and analyzed sweat
• Water, salt, sugar and phosphate (to metabolize
  sugar)
• Later, lemon juice and artificial sweetener to
  make palatable (from wife)
• As interest in the drink spread, Cade offered
  his patent rights to the University
• of Florida. The school turned him down but
  later engaged in a protracted court
• battle over royalty rights and struck a deal
  in 1973.

42
Patents based on biotechnology and human extracts

• USA vs Europe vs Asia
• 1907 First patent of chemical based on human
  extract- adrenaline
• 1923 Insulin
• Organism patent (none before 70s)
• 1972 Chakrabarty/General Electric Co.,
  Pseudomonas metabolizing crude oil
• Diamond vs Chakrabarty genetically engineered
  bacteria, not naturally occurring
• Gene-related patents
• 1993 SmithKline Beecham bought 125M stake in
  Human Genome Sciences Inc.
• Identified 40,000 genes and gene fragments
• Incyte, HGS, Celera, Sequana, etc.

43
Patents based on human extracts and biotechnology

• Patients can hinder the scientific process by
  limiting research and increasing the cost.
• John Moore vs the Regents of the University of
  California
• Hairy-cell leukemia at UCLA Medical Center
  (DGolde)
• Abnormal WBCs bearing hair-like projections
• Potentially fatal form of cancer enlarged spleen
  from 7oz to 22 lbs
• Treatment with chemotherapy
• Splenectomy-gt T-lymphocyte cell line, patented
  1981.
• Spleen cells produced unusual blood protein that
  might be used to develop an anti-cancer agent.
• Court ruled Moore had no rights to profit from
  commercialization of anything developed
• from his discarded body parts.

44
Patents based on human extracts

• Titles of patents Guaymi woman from Panama
• Hagahai man from Paupua New Guinea
• Two dwellers of the Solomon Islands
• Swedish company patented a gene from a person of
  an isolated village in Italy
• 1990s. Panama. Blood samples collected from
  Guaymi people
• Developed cell lines attempted to patent
• Withdrew patent application, Nov06, return cell
  line to the Guaymi
• Cell line still for sale at the ATCC.
• One side
• 2,300 to process a sample vs salary of a Guaymi
  at lt80/yr
• Ethical questions- creating medicines that help
  human beings to avoid suffering, dying
• Another side
• patent a genetic trait of the Guaymi and profit
  from their biological inheritance
• Pat Mooney, Rural Advancement Foundation
  International, 1993
• Guaymi tribe was surprised to discover they
  ed. were invented
• Two American men listed as inventors -gt
  actually, patented a virus
• endogenous virus that stimulates antibody
  production- might be useful in HIV and leukemia
  research

45
Fallout

• Origin stories can clash with DNA data
• threatening a view some indigenous leaders see
  as vital to preserving their culture
• could also jeopardize land rights and other
  benefits based on notion lived in a place
• since the beginning of time.
• What if it turns out youre really Siberian and
  then, oops, your health care is gone?
• Dr D Barrett, co-chairman of the Alaska Area
  Institutional Review Board, sponsored
• by Indian Health Service, a federal agency.
• (SWells) Genographic Project I dont think
  humans at their core are ostriches. Everyone
• has an interest in where they come from.
• But, indigenous leaders point to centuries of
  broken promises.if came from elsewhere, could
• undermine their moral basis for sovereignty
  and chip away at their collective legal claims.
• NGs GP is unlike earlier HG Diversity Project,
  condemned by some groups as biocolonialism
  because
• some scientists may have profited from the
  genetic data that could have been used to develop
  drugs

46
Patents based on plant extracts

• Warfarin, brand name coumadin
• Anticoagulant, as vitamin K antagonist inhibits
  vitK reductase, which recycles oxidized vitK
• UWisconsin, named for Wisconsin Alumni Research
  Foundation
• Synthetic derivative of coumarin, found in many
  plants, esp sweet clover,
• lower levels in licorice and lavender
• Originally developed as rat poison
• 1920s outbreak of previously unrecognized
  disease of cattle in northern US and Canada
• Dying of uncontrollable bleeding from minor
  injuries or drop dead of internal hemorrhage
• with no signs of external injury
• 1921 FSchofield cattle ingesting moldy silage
  from sweet clover, Sweet clover poisoning
• 1940 KPLink and HCampbell at UW anticoagulant
  substance was coumarin
• 1951 attempted suicide recovery medical use
• 1952 registered for use as rodenticide in US
  WARF granted the patent

47
Patents based on plant extracts Development of
chemical synthesis pathways and/or engineered
microbes

• Demand for artificial sweeteners will be over 1B
  by 2010
• Brazzein is 2,000x sweeter than sucrose, tastes
  like sucrose
• 54 AAc, peptide sequenced 1994
• Above, MFariba et al. effects of mutations
• UWisconsin patent on brazzein, a sweetener
  isolated from Cameroon sweet plant Joublie
• USPatent 5326580. Disclosed herein is a protein
  sweetener that has been isolated from
• Pentadiplandra brazzeana Baillon. The sweetener
  is thermostable, lysine rich, and has a
• relative long lasting taste. Also disclosed is
  a recombinant host capable of producing the
• sweetener in large quantities.
• Engineered bacteria to produce brazzein, so
  Camerooneans cannot make money
• selling plant products
• ABerlecBStrukelj, et al. 2006. ApplMicrobiol
  and Biotech. Expression of the sweet-tasting
• plant protein brazzein in E. coli and L.
  lactis a path toward sweet lactic acid bacteria
• Neem tree insecticidal properties, 1995. 29
  foreign patents
• Ecuador Amazonian sacred plant, ayahuasca used
  in traditional healing and visionary rituals

48

Eradicating a species, or two

• Smallpox eradication
• 1956 WHO
• Late 1960s strategy to include mass vaccination
• 1977 last natural case in Somalia
• 1978 medical photographer (et al) near virology
  lab
• 1980 official eradication
• Polio eradication
• On-going

• http//pathmicro.med.sc.edu/lecture/vaccines.htm
• http//www.polioeradication.org/casecount.asp

49

Synthetic genomics

• The Challenge
• Chemical synthesis of life in the lab
• HUrey, SMiller, LOrgel organic chem synthesis
  from inorganics
• Wohlers synthesis of urea, 1828
• Pasteur Spontaneous generation disproved in
  1864
• Khorana Synthesis of 207bp gene for Tyr
  suppressor tRNA in 1979
• Synthesis of self-replicating functional genome
• The Rationalizations
• Basis for understanding minimal cellular life
• Approaches to production of energy,
  pharmaceuticals and textiles
• ex, fixing CO2 from atmosphere to produce
  methane, used for other fuels

50

Chemical synthesis of an infectious virus

• JCello, APaul, EWimmer. Sci02. Poliovirus
  synthesis de novo
• Small non-enveloped RNA virus
• ssRNA at 7,440 nucleotides
• Contains five different macromolecules
• Capsid polypeptides VP1-4 and VPg
• Synthesize with overlapping oligonucleotide
  segments, 400-600 nucleotides
• With VPg replaced by T7 RNA Pol

51

Synthetic Biology synthesis of an infectious
virus

• Synthesis of poliovirus in the absence of a
  natural template
• Oligonucleotide segments (400-600 bases) annealed
  and enzymatically extended, and ligated
• Full-length cDNA is assembled to represent entire
  genetic information of poliovirus as DNA (RNA
  genome)
• cDNA into infectious viral RNA by T7 RNA
  transcriptase
• Seed HeLa cell-free extract replicates to form
  progeny virions
• EWimmer. EMBO Reports. July06

52

Chemical synthesis of an infectious virus

• Entire genome cloned onto a plasmid
• Easier manipulation

53

Chemical synthesis of an infectious virus Prove
it

• Products of in vitro translation and proteolytic
  processing
• HeLa cell-free extract
• 35S methionine-labeled
• control
• wt PV1
• sPV1 cDNA

• Plaque phenotypes
• generated in HeLa cell-free extract
• sPV1 RNA
• wt PV1 RNA

54

Chemical synthesis of an infectious virus

• Biological characterization
• presence and absence of antibody
• polyclonal against types 1 and 2 poliovirus
• PLD50 paralysis or death in 50 inoculated mice
• -gt Results possible to synthesize an
  infectious agent by in vitro chemical-biochemical
  
• means solely by following instructions from a
  written sequence

55
Optimizing synthetic genome construction

• HOSmithJCVenter, et al. PNAS 2003. Generating
  a synthetic genome by whole genome
• Assembly phi X174 bacteriophage from synthetic
  oligonucleotides _at_5,386 bases
• Accurate assembly of 5-6kb genome
• Rapid 14 days start to finish
• Synthetic genome had a lower infectivity than
  natural DNA
• Fully infectious virion recovered after
  electroporation into E. coli
• Propose to assemble larger genomes by joining
  separately assembled 5-6kb genomes
• 60x to give minimal cellular genome lt--
• waited for independent bioethics

56

de novo life minimal genomes

• 1999 Minimal prokaryotic genome
• based on random whole genome transposon
  mutagenesis
• Inactivated one gene per cell
• 300 essential genes for self-replicating
  cellular life described

57

Synthetic genomics bacterium

• CLartigueJCVenter, et al. Sci07 Genome
  transplantation in bacteria changing one
  species to another
• Change Mycoplasma capricolum into Mycoplasma
  mycoides Large Colony (LC)
• Small organisms lacking cell wall
• Antibiotic marker to select for new genome
  de-proteinated new chromosome
• Transplant after several generations, lose old
  phenotype and gains new
• 2D PAGE, protein sequencing blue-linked
  LC-specific Ab stains
• Proof of principles in synthetic genomes
• Ultimate goal of synthetic organisms
• Mary Shelley. 1818. Frankenstein
• http//www.sciencedaily.com/releases/2007/06/07062
  8232413.htm

58

de novo life bacterium

• Artificial organism
• Insights into origins, workings and essence of
  life
• New opportunities to exploit living organisms
• (The Telegraph 6/29/07)

59

Inner life of a Cell

• Multi-disciplinary basic research (many
  fields), recombinant DNA and
• biotechnology, technology (computational and
  technical), and visual arts
• Cellular Visions The Inner Life of a Cell
• What can character animators learn from those who
  render microscopic worlds in 3D? Plenty.
• By Beth Marchant
• July 20, 2006 Source Studio Daily
• The Inner Life of a Cell, an eight-minute
  animation created in NewTek LightWave 3D
• and Adobe After Effects for Harvard biology
  students.
• http//www.studiodaily.com/main/searchlist/6850.ht
  ml