Biotechnology

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6 Δεκ 2012 (πριν από 4 χρόνια και 10 μήνες)

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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



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?image=1081&mode=sequential&flags=0



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/



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/chemistry/articles/berg/index.html



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.”




Frankenfood”

Photo: http://www.cpinternet.com/mbayly/facesofresistance2.htm

http://www.motherjones.com/commentary/power_plays/2000/03/mckibben.html

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…”



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

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



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)



Biodiversity:
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,
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 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




Four Horsemen of the Apocalypse

http://pathmicro.med.sc.edu/lecture/vaccines.htm
Infectious disease vs vaccines
(one approach) ((vs antibiotics))




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

wikipedia

http://images.google.com/imgres?imgurl=http://www.nlm.nih.gov/medlineplus/ency/images/ency/fullsize/17160.jpg&imgrefurl=http://www.nlm.nih.gov/

medlineplus/ency/imagepages/17160.htm&h=320&w=400&sz=18&tbnid=-DFyXPEyexIBTM:&tbnh=99&tbnw=124&prev=/images%3Fq%3Debola%26um

%3D1&start=1&sa=X&oi=images&ct=image&cd=1
Infectious diseases: which is more dangerous/scary?




[[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/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
Complex disease, complex solution



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

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 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


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



Antibiotics biotechnology
QuickTimeª and a
TIFF (LZW) decompressor
are needed to see this picture.

Grace Yim

Graphics: Fan Sozzi



Antibiotics biotechnology

Grace Yim

Graphics: Fan Sozzi

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



Antibiotics biotechnology

Grace Yim

Graphics: Fan Sozzi

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



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

wikipedia



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

wikipedia



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

wikipedia


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


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 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



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
Eradicating a species, or two



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

HeLa cell-free extract

[
35
S] methionine-labeled

control

wt PV1

sPV1 cDNA

Plaque phenotypes

generated in HeLa cell-free extract

sPV1 RNA

wt PV1 RNA



Chemical synthesis of an infectious virus

Biological characterization

presence and absence of antibody

polyclonal against types 1 and 2 poliovirus

PLD
50
= 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”

http://www.sciencedaily.com/releases/2007/06/070628232413.htm



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.

http://www.studiodaily.com/main/searchlist/6850.html