Diagnose Letter Gp Template by ozr14749

VIEWS: 265 PAGES: 59

More Info
									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
•de novo life/Synthetic Genomics
              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

•Photo: http://www.californiacoastline.org/cgi-bin/image.cgi?image=1081&mode=sequential&flags=0               •wikipedia
              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…”

•Photo: http://www.visitasilomar.com/                                                                          •wikipedia
               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
•Photo: http://nobelprize.org/nobel_prizes/chemistry/articles/berg/index.html                                   •wikipedia
                    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 NIH‟s
     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

•“As with the two previous infractions of the NIH rules..” UCSF and Harvard Med…
  “…the UCSD incident posed no threat to public health.”

                                                           •Mar-Apr2000 Mother Jones (Bill McKibben). “Muggles in the Ozone”
                                                           •RE: “Last winter‟s 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/facesofresistance2.htm
                   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
   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
                 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
      •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
                                   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
                               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 “Neptune‟s Medicine Chest)
                   Gold in Yellowstone‟s Microbes

•Yellowstone Park‟s steam vents and Hot Springs
•Unique microbes; extremophiles
•Implications in medicine, agriculture and energy; (basic research), (biotechnology)
•1966 TBrock gathered samples of >80C 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 didn‟t get any money from it. I didn‟t get any money, either, and I‟m not complaining.
  The Taq culture was provided for public research use, and it has given great benefit to mankind.”
                        Harvesting from the oceans,

•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 UC‟s top ten royalty earners @$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
Finding new drugs: Nereus
                         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 commissioner‟s office
•Should be based on medical science, [eg, data and efficacy] not politicized

•Similar worry for Guardisil, 2006

(WAPost 5/25/06)
Infectious diseases, epidemics
                                              Infectious disease vs vaccines
                                              (one approach) ((vs antibiotics))

                                                    •Four Horsemen of the Apocalypse
              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 >170 people
                             •Ebola virus first emerged in 1976, simultaneous outbreaks in Sudan and Zaire
                             •Zoonotic virus; hemorrhagic fever
                             •Four identified strains, Zaire, Sudan, Reston, Cote d‟Ivoire (1 case)
                             •Zaire strain killed 80% (-90%?); Sudan killed 50%
                             •Incubation period of 15 days
                             •No treatments
                                     Complex disease, complex solution

         •[[One is graphic]], one is not- at the beginning
         •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/opt0015.jpg
                                    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 > 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=271778&issue=225; photo: wikipedia
           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 (-> multidrug-resistant, MDR)
•(XDR: extensively drug-resistant)
•Global concerns in the “Jet Age”
•2007: Mexican citizen travels between US and Mexico despite TB diagnosis
                         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”
      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
      •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 world‟s cases)
                   •(reminiscent of “pre-antibiotic era” of 1943)
      •Globally, 4% are MDR
                  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 I‟d come to a country where I‟d 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” … “We‟re 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

•AP 3/15/07 “Hospital worker may have exposed many [532] to TB”
•Including 238 infants (worked in neonatal ICU as well); 260 tested, 3 positives for TB
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
                                       “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/sites/dlc-me/news/ns195dis1.html
                      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 don‟t 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 -> all with multidrug-resistance
     •Return to the “pre-antibiotic era”
                      Antibiotic-resistant staph infection:
                      Mouse model

•10/17/07 WAPost “Drug-resistant Staph germ‟s 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
               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
•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
Antibiotics biotechnology

            QuickTime™ and a
        TIFF (LZW) decomp resso r
     are need ed to see this picture.

                                        •Grace Yim
                                        •Graphics: Fan Sozzi
          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
                   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
                        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
                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
             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 @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
                           Vancomycin: Optimization

•H-MJung…J-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
                           Vancomycin: Optimization

•H-MJung…J-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
                        Vancomycin: Optimization

•H-MJung…J-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
                  “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
                            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
                    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 >$150M 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.”
                   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.
                    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-> 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.”
                            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 <$80/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” -> actually, „patented a virus‟
      •endogenous virus that stimulates antibody production- might be useful in HIV and leukemia research

•Mar95 patent for “the Papua NG HTLV-1” -first human cell line from an indigenous population patented
     •Protests as “patent-free zone.” US dropped patent claim in 96
•European Patent Office to US Co. Biocyte- „ownership of all human blood cells from the umbilical cord‟
     •Of a newborn child (any?) and are being used for therapeutic purposes.
     •Umbilical cord blood (bone marrow). Only isolated blood cells and deep-freeze them; no modifications

•“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 you‟re 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 don‟t 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.”
•NG‟s 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
                          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
                  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 J‟oublie
     •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
     •ABerlec…BStrukelj, 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
                                             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
•http://www.polioeradication.org/casecount.asp         •On-going
                   Synthetic genomics

The Challenge:
•Chemical synthesis of life in the lab
      •HUrey, SMiller, LOrgel: organic chem synthesis from inorganics
•Wohler‟s 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
 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
       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
Chemical synthesis of an infectious virus

    •Entire genome cloned onto a plasmid
    •Easier manipulation
 Chemical synthesis of an infectious virus:
               Prove it…

•Products of in vitro translation and proteolytic processing   •Plaque phenotypes
•HeLa cell-free extract                                        •generated in HeLa cell-free extract
•[35S] methionine-labeled                                      •sPV1 RNA
•control                                                       •wt PV1 RNA
•wt PV1
•sPV1 cDNA
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
• -> Results: “possible to synthesize an infectious agent by in vitro chemical-biochemical
 means solely by following instructions from a written sequence”
Optimizing synthetic genome construction

•HOSmith…JCVenter, et al. PNAS 2003. “Generating a synthetic genome by whole genome
•Assembly: phi X174 bacteriophage from synthetic oligonucleotides” @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 <--
•„waited for independent bioethics‟
   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
                 Synthetic genomics: bacterium

•CLartigue…JCVenter, 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”

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)
                                     “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.

To top