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

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

Thank You!

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