Apply Modern Biotechnology… - U.S. Fish and Wildlife Service

mutebabiesBiotechnology

Dec 6, 2012 (4 years and 9 months ago)

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Apply Modern Biotechnology…


To better
understand and manage

natural
populations:


Molecular genetic tools


Genomics



To
modify or manipulate

organisms:


Repro
-
technologies


Cloning


Genetic engineering



To
determine effects of modified organisms

on
natural populations

Expect combinations
of these

Understand and Manage Natural Populations

Many advantages over older methodologies


Molecular genetic tools


Conservation genetics


Forensics


Pathology


Monitor effects of introduced organisms



Genomics


Understand gene function


Marker
-
assisted selection


Monitor for effects of pollutants, environmental change

Modify or Manipulate Organisms


Repro
-
technologies



*
Chromosome set (ploidy) manipulations



*
Cryopreservation of gametes/embryos


Gynogenesis/Androgenesis


Nuclear transplantation, embryo transfer, etc.



Cloning


propagate endangered species


Somatic cell


e.g., Guar


Primordial cells

e.g. r. trout in Masu salmon
(
Nature
8/5/04)


Embryonic stem cell


Genetic Engineering



recombinant DNA



*
modify performance traits


microbes, plants, animals



control invasive species


Vaccines

*

Already some large
-
scale uses

Oilseed rape not shown.

Data from James 2001 (Int’l Serv. for the Acquisition of Agri
-
biotech Appl. (ISAAA) and USDA NASS April 2004.

0
10
20
30
40
50
60
70
80
90
1996
1997
1998
1999
2000
2001
2002
2003
Percent Adoption
Soybeans
Cotton
Corn
Bt Corn
Crop GEOs In Use

Slide from K. Oberhauser

Examples of Plant GEOs: Field test to Release


Trait
/ structural gene

Species

Drought and salt tolerance
Protein/enzyme genes from bacteria or
other plants

turfgrasses: Bermudagrass,
creeping bentgrass, Kentucky
bluegrass, perennial ryegrasses

Herbicide tolerance, some with altered
growth or disease resistance

CBI or enzyme EPSPS

turfgrasses, poplar,

cottonwood,

Eucalyptus, sweetgum, wheat

Insect resistance

specific
Bt

endotoxin (among many)

Loblolly pine, poplar
,

spruce,
cranberry

Disease resistance

Virus coat protein, various antibacterial
or antifungal genes

papaya
*
, plum

, apple, pear,
grape

*
Deregulated/Commercialized

Release permit CBI = Confidential Business Information

NRC 2004. Biological Confinement of Genetically Engineered Organisms.

http://books.nap.edu

Plant GEOs: Research to Field Tests


Trait
/ structural gene

Species

Decreased lignin content

Specific enzymes from bacteria or poplar; ligase
antisense gene from poplar; or CBI


poplar
#
, pine
#
,
turfgrass (
Paspalum
notatum
)
#


Bioremediation

Mercuric ion reductase (from E. coli), cytochrome
P450 (from human)

poplar
#

Pharmaceuticals, biologics, industrial chemicals

swine viral vaccine, avidin, trypsin, high laurate
many others in research

corn
-

swine vaccine
#
,
avidin
**
, trypsin
*
,
canola
-

laurate
*

numerous other crops

Rehabilitate endangered species

Fungal blight resistance genes from Asian chestnut

American chestnut

#

Field test
*
Commercialized
**
Commercialized via field test notification

NRC 2004 Biological Confinement of Genetically Engineered Organisms. Snow et al. 2004 Ecol. Soc. Am. GEO
Position Statement. Nature Biotechnology 2004 Editorial.

www.glofish.com

GloFish casts light on murky

policing of transgenic animals

Nature 27 November 2003

The New York Times Nov 22, 2003

Gene
-
Altering Revolution

Nears the Pet Store:

Glow
-
in
-
the
-
Dark Fish

First Transgenic Animal on U.S. Market

Marketed without regulatory environmental review. FDA is lead authority.

Could become
first transgenic
animal approved
for large
-
scale
farming and
human food

The New York Times

Age = 7.5 months

Transgenic = 1.2 Kg., Unmodified = 200 g

Http://webhost.avin.net/afprotein/peidof.htm

Engineered with ocean pout
antifreeze gene promoter +
chinook salmon growth
hormone gene



Novel proteins or
novel gene regulation

alter
physiology. Alter ecological roles?

(Devlin et al. 1994)

Growth hormone expressed
in cold waters & unlinked
from seasonal temp. cue.

Smoltification precocious.

Age =14 months

Largest transgenic


= 41.8 cm

Age = 6 months; autotransgenic = 255.4 g (Nam et al. 2001)

Auto
-
transgenic
mud loach:
β
-
actin promoter
linked to GH
gene. Growth
increase >30
fold.

Gigantism.

Aquatic GEOs in the Pipeline

Marine Biotechnology Briefs

http://www.fw.umn.edu/isees/MarineBrief

Enhanced
growth
&

food conversion

growth hormone genes

Atlantic salmon
*
,

coho salmon,
common carp
#
,

mud loach,

Nile tilapia
#
,

rainbow trout
*



Disease resistance

mammalian interferon

moth
or porcine

antibacterial

cecropin

B


grass carp
,

channel catfish,

medaka


Cold water
tolerance

ocean pout
a
ntifreeze protein


goldfish


C
arbohydrate digestibility

mammalian
enzymes
for glucose
metabolism



rainbow trout








Trait

/
structural gene

Species



*
applying for approval: U.S., Canada.
#

preparing to apply: Cuba, P.R. China

Aquatic GEOs in the Pipeline

Marine Biotechnology Briefs

http://www.fw.umn.edu/isees/MarineBrief



B
ioremediation

chicken metallothionein

(heavy metals binding)


unicellular
algae
(
Chlamydomonas
)



Thrive in absence of light

h
uman glucose transporters


microalgae

normally
oblig
a
te photoautotroph


Secrete pharmaceutical

human clotting factor VII


N
ile tilapia


R
etroviral vectors

with marker genes

s
tep towards engineering
production traits


live bearing fish
(
Poeciliopsis
), crustacean
(crayfish),

mollusk (surfclam)



Trait

/
structural gene

Species



Various genetic engineering methods


2 examples



Sex Ratio Distortion



daughterless carp technology



Engineered fitness disadvantage



site
-
specific selfish gene




Feasibility study for FWS


genetic biocontrol of invasive fish in
Gila River Basin, AZ, focus on green sunfish, red shiner, mosquito
fish (Kapuscinski et al., ongoing)

Deleterious transgene spread


to control invasive fish species

www.marine.csiro.au/LeafletsFolder/pdfsheets/Daughterless_carp_thirteenmay02.pdf

Mallee Research

Station

4 nights’ catch, 1917

Lascelles Victoria

Genetic Biocontrol

House mouse plagues

Slide from Tony Peacock

Virally
-
vectored
immunocontraception

Isolate ZP3

DNA

Insert DNA into

mouse
-
specific virus

(recMCMV)

Infect mice with recMCMV

The mouse’s immune

system blocks reproduction

The egg protein ZP3 is

essential for reproduction

Slide from Tony Peacock

Risk Assessment and Management


General agreement on case
-
by
-
case
approach for GEOs



Environmental biosafety science develops
methodologies and generates empirical data
needed for scientifically reliable risk
assessment and management


Strategies to cope with limits to prediction

Systematic Risk Assessment

1. Identify hazard

-

what event posing harmful
consequences could occur?
[knowledge is best here]


2. Estimate exposure
-

how likely is the hazard?
[ability varies case
-
by
-
case; e.g. lack confirmed
methodology for fish]


3. Predict harms & severity
-

what would be
harms and how bad are they?
[ability varies; need
confirmed methodology]

4. Estimate risk


likelihood versus severity of harm
[limits to quantification; depends on prior steps]



Kapuscinski 2002. Controversies in Designing Useful Ecological Assessments….

National Research Council (NRC) 2004. Biological Confinement of Genetically Engineered Organism


Systematic Risk Management

Risk reduction
-

what can be done to reduce
likelihood or mitigate consequences of harm?
[Focus
has been on confinement


see NRC 2004]


Post
-
release monitoring
*

-

how effective are risk
reduction actions?
[Little attention so far]


Remedial action

-

What corrective action if
monitoring findings are unacceptable?
[Largely
ignored so far]


*only way to learn and improve future decisions (Adaptive
Management)


Kapuscinski 2002; NRC 2004

1. Identify potential hazards



Gene flow to related taxa (interbreeding)


Invasion by alien species (is GEO more invasive than
unmodified?)


Interact with non
-
target organism


Evolution of resistance (pesticide
-
producing GEO)


Changes in viral disease (virus
-
resistant GEO)


Horizontal gene flow (1
arily

microorganisms)


Scientists’ Working Group on Biosafety 1998.
www.edmonds
-
institute.org/manual.html

Pew Initiative on Food and Biotechnology 2003

National Research Council 2004

Ecological Society of America. 2004

2. Estimate exposure to hazard


Need a confirmed methodology involving
tractable and repeatable tests that can be
conducted in confined settings



Don’t have this yet but…



Net fitness methodology is one promising
candidate

(Net fitness methodology: Muir and Howard 2001, 2002)

wild
-
type medaka

transgenic medaka

Photos: Mike Morton

top view

Can we confirm the methodology? Ongoing test …

side views

Kapuscinski laboratory

0.0000
0.0250
0.0500
0.0750
0.1000
0.1250
400 (a)
400 (b)
400 (c)
67 (a)
67 (b)
67 (c)
treatment (sGH line number)
transgene frequency
initial freq
final freq
0.0000
0.0250
0.0500
0.0750
0.1000
0.1250
400 (a)
400 (b)
400 (c)
67 (a)
67 (b)
67 (c)
treatment (sGH line number)
transgene frequency
initial freq
final freq
Control


x 3 replicates

1
st

GEO line


x 3 replicates

2
nd

GEO line


x 3 replicates

unmodified
population (N = 353)

Released 20
pMTsGH
-
400
into unmodified (N=353)

Released 20
pMTsGH
-
67


into unmodified (N=353)

1
st

trial: transgene fate
after 2 generations;
population size equal
across all treatments at
end of trial.



(2
nd

trial currently
underway.)


[Method predicts
transgene spread]

[Method predicts
transgene spread, then
population decline]

3. Predict Harms and Severity



Examples for fish and wildlife:


1.
Loss of unique genetic resources


e.g., center of
origin, extinction by hybridization

2.
Decline in abundance of species of special
concern
-

target of fishing/hunting, endangered,
keystone in food web, culturally important, etc.

3.
Decline in resilience of biological community

ability to recover from external disturbances


Challenges: cumulative, long
-
term, large
-
scale



Scientists’ Working Group on Biosafety 1998. Pew Initiative on Food and Biotechnology 2003

Predicting harms:

transgenic fish for aquaculture

(photo: Devlin et al. 1994)



Guidance based on literature syntheses, but few GEO studies



L
ab study of one line of growth
-
hormone transgenic coho
salmon (Devlin et al. 2004)


relevance to field conditions?

High food availability:

transgenics did not
competitively interfere
with growth of
nontransgenics.


Low food availability:

populations with
transgenics crashed and
those without continued
to increase in biomass.

UCS 2002. Pharm and Industrial Crops

For pharma/industrial crops:



Potential harm to wildlife feeding on plants / seeds



Potential harm to ecological resilience



via exposure of pollinators, herbivores, soil inhabitants



via transgene spread to wild/weedy relatives



via bioaccumulation

Industry
Projections:

Market of
$100s
billions by
2010

Hazard

Potential Harm

Density
-
dependent compensation
for X years

Wipe out endangered fish before
biocontrol effect prevails

Failure in intended trait change

Increased number of fit non
-
natives
increases disruption of native fish

Transgene side effect on trait that
enhances predation or
competition

Increases disruption of native fish before
biocontrol effect prevails

Transgene spread to native range
of species

Depress or extirpate native populations

Transgenic fish caught for eating

Harm to human health

Horizontal gene transfer to non
-
target species (very hypothetical)

Depress populations of non
-
target species

Transgenic Fish for Biocontrol

Decreasing likelihood (in general)

Kapuscinski et al. In preparation

Photo: Nick Didlick

Holmlund & Hammer (1999)


Individual species


Equilibrium


Linear


gradual
change


PREDICTABLE


Socio
-
ecological
system


Multiple states


Non
-
linear


can flip to
new state


EXPECT SURPRISE

Prevailing Framework

Emerging Framework

Functional homogenization

reduces resilience

Composition of,
variation in, and
spatial distribution
of traits of the
species in a
community.


A
-
C
: historical
communities.

a
-
c
: homogenized
communities.

External


shock

Olden et al. (2004)

Biotechnology
-

Prevailing Approach

Small polygon



policy decision

Green dotted arrow



ad
-
hoc

learning

Expert Priority
Setting
Benefits
Assessment
Field Test of
Desired Traits
Risk
Assessment
and
Management
Ad-Hoc
Detection of
Problems
Corrective
Actions
Field
Testing
Decision
Commer-
cialization
Decision
Policy
Evaluation
& Review
Research
Funding
Decision
TIMELINE FOR PREVAILING RESEARCH, DEVELOPMENT AND APPLICATION
Research &
Development
Studies
Risk
Assessment of
Proposed
Field Test
Deliberation usually entails public review just before or after decision

Biotechnology
-

Pro
-
Active Approach

Small polygon



policy decision.
Solid green arrow



adaptive learning.
Italics



pro
-
active steps

Participatory
Priority Setting
&
Safety Criteria
Setting
Prospective
Benefits
Assessment
Prospective
Risk
Assessment
Safety Design
Benefits
Assessment
Field Test of
Desired Trait

& Safety
Safety
Management
Participatory
Benefits &
Safety
M
onitoring
and Follow-up
Long-term,
Large Scale
Studies
Field
Testing
Decision
Commer-
cialization
Decision
Policy
Evaluation
& Review
Research
Funding
Decision
TIMELINE FOR PROACTIVE RESEARCH, DEVELOPMENT AND APPLICATION
Research &
Development
Studies
Safety
Testing
Risk
Assessment of
Proposed
Field Test
Kapuscinski et al. 2003. Nature Biotechnology

Deliberation is linked to analysis from the outset

Prevailing approach


little steering to be safer
from outset

Risk (likelihood of harm)
High
Low
Severity of Harm
Low
High
NRC 2004. Miller et al. 2002

Risk (Likelihood of Harm)
Frequent
Very close
to never
Severity of Harm
None
As bad as
it can get
Likelihood should decrease as
severity of harm increases.
‘Safety first’: safety criteria to impose upper limit to risk

©

ISEES & S. Hann 2003

As bad as
it can get
Very
large
Sig-
nificant
Small
None
Severity of Harm
(Includes Cumulative Effects)
Frequent
Common
Rare
Very close
to never
Very unlikely
Maximum Acceptable
Likelihood of Harm
Safe
enough
Not safe
enough
© ISEES & S. Hann 2003

Example of Deliberation Point: Effect
of Benefits on Upper limit of
Acceptable Risk
As bad as
it can get
Very
large
Sig-
nificant
Small
None
Severity of Harm
Frequent
Common
Rare
Very close
to never
Very unlikely
Maximum Acceptable
Likelihood of Harm
Increasing
Benefits
Decreasing
Benefits
© ISEES & S. Hann 2003

Pro
-
active Australian approach:

Genetic biocontrol of invasive fish


Will the genetic method work?


Under real conditions


Credible evidence before deployment



What are the risks?


Environmental


Human health



Answer via multi
-
prong program



Progress from simple to more complex
tests of efficacy and potential risks


Parallel components

… Gila Basin feasibility study will
advise go/no go points for:

1.
Development of genetic methods

2.
Efficacy testing

3.
Modeling


to inform components
1, 2 & 5

4.
Target species ecology


to inform
2, 3 & 5

5.
Risk analysis

6.
Community/public awareness and involvement

with links to
5, 7 & 8

7.
Seeking regulatory approval

8.
Post
-
approval monitoring


to verify
2 & 5

Pro
-
active Example: Safety First Initiative

2001


Public workshop obtained extensive feedback on approach

2002


U.S. public
-
private coalition: Safety First Initiative Executive
Advisory Board and Steering Committee

2003


Kapuscinski et al. Nature Biotechnology 21(6):599
-
601

Propose cross
-
sectoral working groups to develop safety standards.
Partners welcome.

Reports at www.fw.umn.edu/isees

Possible Bureau Roles
-

Science


Support research and outreach


inform more pro
-
active approach


scientific analysis


involve ecologists, conservation geneticists, etc.


representative deliberation


Provide biosafety research sites


confined field tests


contained labs for fish & other aquatics


Enhance species and ecological baselines


pre
-
commercialization studies


post
-
commercialization monitoring and verification tests


Long
-
term, large ecosystem scales

‘Coordinated Framework’ for


Regulating Biotechnology


Food and Drug Administration (FDA)
claims regulatory lead over transgenic
animals, including fish



Drug regulations forbid public review



FDA lacks expertise &
mandate

for F&W



FWS & NMFS can stop only if harms to
threatened or endangered species

Federal Regulation
-

Uncertainties


FDA explicitly did not regulate the GloFish:




In the absence of a clear risk to the public
health, the FDA finds no reason to regulate
these particular fish
.” (FDA Statement
released Dec 9, 2003)


Where does this leave regulation of
environmental safety?


Authority over biocontrol transgenic animals
that are not eaten by humans


such as red
shiner, nutria?



Possible Bureau Roles


Resource Management


Larger role in regulation


biotechnology applied to F&W & natural ecosystems


transgenic fish regulation is a pressing need


Options: from formal MOU with lead agency to
establishing lead authority


Restore transparency of review (NEPA, ESA)



Establish policies & procedures/standards


GEOs on federal lands


Commenting on other agency actions



Develop federal GEO monitoring program


tracking spread in the environment


detect unwanted/unexpected problems


safety verification testing

Decision

Implementation

Evaluation

Public

Officials

Natural

Scientists

(few disciplines)

Analysis

Define

Problems

Select

Options

Information

Gathering

Synthesis

National Research Council. 1996. Understanding Risk

Public Comment

Dominant Risk Decision Process

Public Demand

Decision

Learning and Feedback

Implementation

Evaluation

Analysis

Deliberation

Public

Officials

Natural &

Social

Scientists

Interested


and

Affected Parties

Analysis

Deliberation

Define

Problems

Select

Options

Information

Gathering

Synthesis

National Research Council. 1996.



Adaptive management approach


“An open process wins every time.”

Stu Hann

Adaptive Biosafety Assessment & Management


Set

Goals


safe use of

GEOs

Information
base

Implementation

release, permits

risk management

Monitoring

mark GEOs,

databases

Problem

Analysis

all R & D phases

Policy Design

assess risks

identify choices

Kapuscinski et al. 1999

Risk Assessment


(or safety verification)

At present, for
most

transgenic fish: It is
very difficult to
conduct a reliable lab
or confined field test
to determine, ahead
of time, what is the
severity

of the
environmental harm.
However….

As bad as
it can get
Very
large
Sig-
nificant
Small
None
Severity of Harm
(Includes Cumulative Effects)
Frequent
Common
Rare
Very close
to never
Very unlikely
Maximum Acceptable
Likelihood of Harm
Safe
enough
Not safe
enough
It is easier to to
determine, ahead of
time, the
likelihood

of
environmental harm
by a transgenic fish:



Net fitness
methodology



Integrated
confinement system



As bad as
it can get
Very
large
Sig-
nificant
Small
None
Severity of Harm
(Includes Cumulative Effects)
Frequent
Common
Rare
Very close
to never
Very unlikely
Maximum Acceptable
Likelihood of Harm
Safe
enough
Not safe
enough
If the net fitness of the
genetically engineered
line fits the
Purging
Scenario
.


(If purging in lab test,
then purging also
likely in more hostile
natural environment.)

As bad as
it can get
Very
large
Sig-
nificant
Small
None
Severity of Harm
(Includes Cumulative Effects)
Frequent
Common
Rare
Very close
to never
Very unlikely
Maximum Acceptable
Likelihood of Harm
Safe
enough
Not safe
enough
Stringency of
integrated
confinement system
should reflect
predicted risk and
severity of harm.
Example: high
stringency
confinement to
achieve very low risk
if severity is very
large.

As bad as
it can get
Very
large
Sig-
nificant
Small
None
Severity of Harm
(Includes Cumulative Effects)
Frequent
Common
Rare
Very close
to never
Very unlikely
Maximum Acceptable
Likelihood of Harm
Safe
enough
Not safe
enough
Hazard scenario determines harms to assess

HAZARD SCENARIO

ASSESS ECOLOGICAL CONSEQUENCES

gene

flow to

wild

relatives

Net fitness
differences between
GEO and wild or
feral relatives

Considered
safe

Alter
genetic
diversity
?

Harm
species of
special
concern?

Reduce
community
resilience?*

Purging

GEO < wild or feral

assess


assess





Spread

GEO ≥ wild or feral



assess

assess

assess

Trojan gene

Opposing traits =

population decline



assess

assess

assess

increasing difficulty

* Resilience could be key question under widespread use of aquatic GEOs

Hazard scenario determines harms to assess

HAZARD SCENARIO

ASSESS ECOLOGICAL CONSEQUENCES

Alien
species
invasion

Net fitness
differences between
GEO and wild
-
type
alien species

Considered
safe

Alter
genetic
diversity
?

Harm
species of
special
concern?

Reduce
community
resilience?

Disappearance

GEO < wild
-
type

assess


assess





Establishment

GEO ≥ wild
-
type



assess

assess

Effective
Establishment

Repeated entries



assess

assess

increasing difficulty

growth
enhanced

Age at
maturity

Juvenile

Viability

Mating

Success


Fecund.

Male

Fertility

Adult

Viability


Scenario

r. trout

wild strain

Devlin et al. 2001

+?



amount n/a

+

37
-
83 times
larger

?

?

?


candidate

coho

Devlin et al. 1994

+ ?

?

early smolt

+ ?


?

?

?


for

coho

Devlin et al. 1995

+ ?


?

early smolt

+ ?

?

?

?

Spread

Nile tilapia

Rahman &
Maclean 1999

?

?

+

3 times
larger

?



zero to low

?

or

mud loach

(huge)

Nam et al. 2001

+ ?

likely very
early

= ?

yolk
-
sac
absorption

+ ?

?

=

?

Trojan

Gene?

medaka

Muir & Howard
2001

+

12.5%
earlier



30% lower

=

+

29%
greater

=

=

Spread

predicted

Net fitness data missing for most transgenic fish

Decreasing influence of trait on net fitness

Pro
-
active approach
example

Involving experts,
affected parties, and
public at large at key
points.


Multi
-
stakeholder
workshop far ahead of
possible GE fish
introduction in Thailand.

Photos: Mike Morton

NRC 1996. Understanding Risk: Informing
Decisions in a Democratic Society

Build higher dikes
to resist floods

Multi
-
layer barriers
for effluent from
pond drain

Physical
confinement
-

examples

Recirculating Aquaculture


Systems


Three Legs of Biotechnology Governance




Government


Regulations based
on reliable safety
science


Safety professional
certification


Producers

(businesses & public institutions)


GEO & product safety standards


Safety leadership
-
top mgm’t to
certified safety professionals

Public


Safety research


Safety education and
training


Safety deliberation