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Risk assessment and regulation of RNAi Pesticides in the context of GE plants and the regulation of novel plant breeding techniques in the United States
Andrew F. Roberts, Ph.D. Deputy Director, CERA September 19, 2013
Contents of the talk
What is RNAi? Molecular mechanism
Applications in GE plants
Review of the ERA Paradigm
Conference: Problem Formulation for the ERA of RNAi Plants
Objective and approach
Case Studies
Results of the conference
Implications for Regulation of New Plant Breeding Techniques in the USA
What is RNAi?
Set of processes mediated by small regulatory RNAs Sequence-specific RNA degradation Some are inducible responses to defend against invasive nucleic acids (e.g. defense mechanisms in plants against plant viruses) Some are endogenous pathways used to control gene expression
Credit: V. Vance
RNAi is an important biological
phenomenon
Ancient pathways present in virtually all eukaryotic organisms
Different names for the same thing: Plants - post-transcriptional gene silencing (PTGS)
Fungi - quelling
Animals - RNA interference (RNAi)
Silencing pathways share genetic requirements and biochemical features
Triggered by double stranded RNA (dsRNA)
Credit: V. Vance
Why is RNAi useful to plant
breeders?
A powerful genetic tool
Can be induced experimentally using transgene constructs that make dsRNA
Once silencing is triggered, any RNA with homology to the target RNA is destroyed
Major biotechnology implications
A way to turn off gene expression in a sequence-specific way
Credit: V. Vance
Applications of RNAi in the plant
sciences
Example transgenic RNAi traits in development
Modified oil composition
Potato starch composition (e.g., Amflora)
Decaffeinated coffee
Reduced lignin alfalfa
Increased essential amino acids in corn
Nematode resistant soybean
Reduced phytate sorghum
Credit: G. Heck
Approved (presumptive) RNAi plants Event Crop Trait ERA Approval (Country, Year)
FlavrSavr Tomato Delayed softening USA (1992) Mexico (1995) Japan (1996)
ZW-20 Squash Virus resistance USA (1994)
CZW-3 Squash Virus resistance USA (1996)
55-1/63-1 Papaya Virus resistance USA (1996)
RBMT15-101, SEMT15-02, SEMT15-15
Potato Virus resistance (+ Bt)
USA (1999) Canada (1999)
RBMT21-129, RBMT21-350, RBMT22-082
Potato Virus resistance (+Bt)
USA (1998) Canada (1999)
Vector 21-41 Tobacco Reduced nicotine USA (2002)
C5 Plum Virus resistance
USA (2007)
X17-2 Papaya Virus resistance
USA (2009)
DP-305423 Soybean Modified seed fatty acid content
Canada (2009) USA (2010)
A brief explanation of the generalized
ERA paradigm for GE plants
Comparative Assessment Conducted on a case by case basis Considers Characteristics of the Plant, Introduced Trait and Receiving Environment
As well as their interactions
Makes use of the concept of Familiarity
Plant
Trait Environment
There is no international standard for
ERA of GE plants, but…
Countries consider similar broad protection goals and potential for harm, including:
Potential for the GE plant to be a weed of agriculture or invasive of natural habitats Potential for gene flow to related species that might become weeds of agriculture or invasive of natural habitats Potential to adversely impact non-target organisms or biodiversity
The types of data collected to address these are also generally similar:
Phenotypic characterization/comparison Laboratory toxicology / feeding studies Observations from field trials
Differences in decision making typically stem from Differences in tolerance to risk and uncertainty Differences at the Policy/Political level NOT fundamental differences in ERA methodology
Conference: Problem Formulation for
the ERA of RNAi Plants
June 1-3, 2011 in Washington, D.C.
Organized by CERA
Funded by the National Institute for Food and Agriculture (NIFA), USDA
Biotechnology Risk Assessment Research Grants Program (BRAG)
Tri-partite conference organizing committee
Conference of 40 scientists: regulators, academics, private sector, NGOs
Why?
RNAi represents a substantially different technology than “traditional” genetic engineering (i.e. the introduction of genes encoding proteins which mediate phenotype)
No protein produced
Highly dependent on nucleic acid sequence
May or may not be a “donor organism”
It is important to have a deliberate examination of how the ERA paradigm being used for GE plants can be applied to new technologies
RNAi conference objectives
To share information about current applications of RNA interference to produce novel, transgenic plants
To explore if problem formulation for RNAi plants leads to new or additional risk hypotheses when compared with non-RNAi plants expressing similar traits
Scientific exchange – not a policy discussion
Conference focused on four case
studies
Participants were asked to: Identifying risk scenarios – whereby the introduction of a GE plant with an RNAi trait might have an adverse impact on a protection goal
Identifying testable hypotheses related to risk scenarios
Case Studies Insect resistant maize
Nematode resistant soybean
Nutritionally enhanced sorghum
Reduced allergen soybean
Relevant Protection Goals
USEPA: No unreasonable adverse effects upon man or the environment
No non-target organism affected No gene flow leading to enhanced weediness and altered exposure scenario No environmental fate leading to altered exposure scenario
USDA APHIS: No potential risks to agriculture and the environment
No increase in disease and pest susceptibilities No increase in weediness characteristics No increase in weediness of sexually compatible plants No increase in harm to other organisms (beneficial, threatened and endangered species) No plant pest effects from changes in cultivation practices
Case Study 1: Insect-resistant RNAi
maize
Credit: J. Masucci
November 2007, Volume 25, pp. 1187 - 1328
Plant Cell Corn Rootworm Cell
Stable IR transgene
target gene
mRNA recognition 5’cap AAAAA
target gene
Dicer
21-24mers
RISC
21-24mers
cleavage
Ingestion (uptake) Dicer
dsRNA
dsRNA
Case Study 2: Soybean Cyst
Nematode Resistant Soybean
Currently under development by USDA Agricultural Research Service
Soybean Cyst Nematode The major pest of soybean in the U.S.
No chemical control available (legal and effective)
Goal Develop soybean resistant to all SCN genotypes (and other nematodes) using RNAi gene silencing
Approaches Turn off critical nematode genes and proteins important to nematode survival using RNAi targeted to nematode genes with essential functions
Credit: B. Matthews
Case Study 3: Nutritionally Enhanced
Sorghum
Reduced-phytate (phytic acid)
• Phosphorous storage complex
• Binds zinc and iron
• Decreases bioavailability
http://coolinginflammation.blogspot.com/2009_06_01_archive.html
Credit: J. Anderson
Source: Buchanan et al., Biochemistry and Molecular Biology of Plants. 2000
One approach to
reduce phytate
is through RNAi
suppression of the
myo-inositol
kinase gene
X
Reduced
Credit: J. Anderson
Nutritionally Enhanced Sorghum
Case Study 4: Reduced Allergen
Content Soybean
Soybean contains many allergens
P34 is the primary human neonatal allergen
Transgenic Soy has been produced using RNAi to eliminate P34 protein
Credit: E. Herman
Agglutinin
Glycinin
A5A4B3
Basic
7S globulin
Kunitz TI
Napin-type
2S albumin
Dehydrin
Glycinin
basic chain
Sucrose binding
protein
Alpha prime
beta-conglycinin
Lipoxygenase
Gly m Bd 30k/P34
Alpha
beta-conglycinin
Beta
beta-conglycinin
Glycinin
acidic chain
What did we do?
Participants were divided into four breakout groups
Working independently
Each group was asked to work on two of the four case studies
Risk Scenarios (pathway to harm) Risk Hypotheses Information/Data that would be useful to corroborate or refute the hypotheses
The work of two groups were then combined for each case study Plenary then reconvened to go over the results and identify points of consensus
Points of consensus from the RNAi
conference
The paradigm currently applied to the environmental risk assessment of genetically engineered plants is adequate for the assessment of RNAi plants.
This Paradigm is Robust and Broadly
Applicable
Comparative Assessment Conducted on a case by case basis Considers Characteristics of the Plant, Introduced Trait and Receiving Environment
As well as their interactions
Makes use of the concept of Familiarity
Plant
Trait Environment
Points of consensus from the RNAi
conference
No plausible risk hypotheses were identified that can be considered unique to RNAi mechanisms when compared to other GE plants with similar traits.
The same tests and protocols that are used for evaluating other GE plants will be sufficient for testing RNAi plants, including plants expressing pesticidal traits.
Points of consensus from the RNAi
conference
The use of RNAi technologies allows for the use of alternative informative tests, such as bioinformatic analyses, to address certain risk questions. For plants expressing pesticidal dsRNAs, bioinformatics can be applied to characterize potential susceptibility of relevant NTO species. The accumulation of bioinformatic data that defines thresholds for activity spectra based on shared sequence identity will reduce the need for NTO testing.
Points of consensus from the RNAi
conference
Baseline data about environmental fate of dsRNA will be broadly useful for future exposure analyses
Implications for Regulation of New
Breeding Techniques in the USA
Coordinated Framework Policy Statement – U.S. agencies will regulate the products of biotechnology in accordance with their authorities under existing safety regulations
USDA Plant Protection Act
FDA Federal Food Drug and Cosmetic Act (FFDCA)
US EPA Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) FFDCA (Pesticide residues in food)
USDA Regulation of New Plant
Breeding Techniques
Plant Protection Act
Regulations (7 CFR 340) Defines “plant pest”
A “regulated article” must meet two requirements
Produced using genetic engineering (recombinant DNA techniques)
– AND
Donor organism, recipient organism, vector, vector agent, is a plant pest
– OR
Is an unclassified organism the Administrator determines is a plant pest or has reason to believe is a plant pest
US EPA
FIFRA Regulates the use of pesticides through a “registration” process
“Plant incorporated protectants” (PIPs) are considered pesticides
Bt proteins
Virus resistance (RNAi mediated)
EPA regulates the pesticide, not the plant
EPA will be holding a Scientific Advisory Panel meeting to discuss RNAi pesticides in October
US FDA
Regulates food safety
Primarily post-market safety authority
“Novel” foods are subject to a voluntary consultation process (premarket)
Consultation is voluntary
Safety is mandatory
The U.S. will continue regulating
under the Coordinated Framework
For RNAi Plants
USDA will likely regulate RNAi plants with sequences from “pests”
US EPA will regulate any pesticidal RNAs
FDA will continue to have oversight over food safety
“voluntary” consultation process is likely to be requested by developers of GE plants with RNAi mediated phenotypes
Acknowledgements
Slides from: Vicki Vance - University of South Carolina Greg Heck – Monsanto Company Alan Gray - Centre for Ecology and Hydrology, UK Jim Musucci – Monsanto Company Ben Matthews – USDA ARS Jennifer Anderson – Pioneer Eliot Herman – (Danforth Center) University of Arizona www.cera-gmc.org
Developments since the conference
Zhang et el Report evidence that micro RNA (miRNA) from rice can be found in human and mouse tissue
Evidence suggesting that this miRNA might regulate gene transcription both in vitro in human cells and in vivo in mice
This result is surprising, and captured a lot of attention
But does it impact the results of the conference?
I think the answer is no
Risk Hypothesis for Mammalian
Consumption of dsRNA
Risk Scenario Consumption of dsRNA in food leads to harm (death or illness)
The mechanism may be different than for GE plants expressing protein, but the assessment endpoint is the same
Similar to pesticides with different mode of action
Further, the same tests currently used to assess GE plants should be sufficient to address risk from an RNAi plant
Toxicity testing (dsRNA) Feeding studies
Verbatim Consensus Points
The paradigm currently applied to the environmental risk assessment of genetically engineered plants is adequate for the assessment of RNAi plants. No plausible risk hypotheses were identified that can be considered unique to RNAi mechanisms when compared to other GE plants with similar traits. The same tests and protocols that are used for evaluating other GE plants will be sufficient for testing RNAi plants, including plants expressing pesticidal traits. The use of RNAi technologies allows for the use of alternative informative tests, such as bioinformatic analyses, to address certain risk questions. For plants expressing pesticidal dsRNAs, bioinformatics can be applied to characterize potential susceptibility of relevant NTO species. The accumulation of bioinformatic data that defines thresholds for activity spectra based on shared sequence identity will reduce the need for NTO testing. Baseline data about environmental fate of dsRNA will be broadly useful for future exposure analyses