Topical Application of dsRNA for Pest Management · Flavr Savr tomato Virus resistance Soybean oil composition Quality Traits Calgene 1994/1995 (USA, CAN, MEX) • Squash 1994 (USA,

Monsanto Company Confidential

Topical Application of dsRNA for Pest Management

Greg Watson, PhD Monsanto Chemistry and Seed Regulatory SFIREG Policy and Other Matters Meeting September 20, 2016


Today’s Goals and Proposed Discussion Outline

Goals Engage with state regulators to initiate a dialogue Outline Basics of RNAi and potential for dsRNA-based products

Overview - RNA history of safe use: dietary safety, environmental fate, and ecological safety •  Examples of approach to inform problem formulation and risk

assessment

Short introduction to the Bee Health Varroa and Canola Flea Beetle projects

2


BioDirect ™ is Based on New Applications of dsRNA-based Technology

RNAi is a Natural Mechanism to Control the Expression of a Gene

DNA

Gen

e

RNA

Protein Produced

DNA

Gen

e

RNA

“Transfers information from gene to construct a protein”

Cells make small RNAs that specifically target

mRNA

RNA broken up Protein

Protein production reduced

“Oligo”

3

dsRNA


PlantproducestheRNA

TworoutestouseofdsRNA-basedproductsincropimprovementTopically

applytheRNA

4

Leading BioDirect™ projects: Varroa mite / managed honey bees Canola Flea Beetle

Corn Rootworm 3 (CRW3) Expresses DvSnf7 RNA

BioDirect™ projects in early stages of

Development - small scale efficacy testing to determine

potential for pest control


Regulatory Considerations for BioDirect • RNA-based suppression is a naturally occurring process, and is the basis for many desired phenotypes

• RNA-based technologies offer new modes of action in managing hard to control pests and viruses

• RNA has a history of safe consumption

• RNA offers specific gene suppression that greatly reduces potential impact on non-target organisms (NTOs)

• The current safety assessment strategy for exogenously applied RNA-based products is based on the same safety assessment paradigm used for other biological products

RNA is in food

Natural process

Favorable NTO profile

Existing paradigm is adequate

Additional tool

5


Ø  Core guideline studies to demonstrate lack of hazard Ø  Ecological problem formulation and use of data / risk

assessment framework

The existing paradigm for dietary and environmental safety is robust and utilizes a number of endpoints to evaluate product safety:

Existing Regulatory Paradigm for Biopesticides is Appropriate for dsRNA based products

‘Consistent with section 408(b)(2)(D) of the FFDCA, EPA has reviewed the available scientific data and other relevant

information in support of this action and considered its validity, completeness and reliability and the relationship of this

information to human risk. EPA has also considered available information concerning the variability of the sensitivities of

major identifiable subgroups of consumers, including infants and children. The following information was used in a

weight of the evidence approach to show the absence of any dietary hazard associated with

DvSnf7 RNA.’ EPA Attachment #5, SAP Public Docket

CRW3 Human Health Assessment August 16, 2016 6


RNAi-based traits are common in nature, not new to agriculture

Tutejaetal.(2004)PlantCell16:19-35

Kosekietal.(2005)PlantCellPhysiol.46:1879-1883

DellaVedovaetal.(2005)GeneMcs170:1989-2002

Chalcone Synthase Suppression: •  Soybean seed color •  Maize stalk color •  Star pattern in petunias

RNAi Mediates Other Natural Phenotypes: •  Low glutelin rice •  Viral and pathogen resistance

7


Flavr Savr™ tomato Virus resistance Soybean oil

composition Quality Traits

Calgene 1994/1995

(USA, CAN, MEX)

•  Squash 1994 (USA, CAN) •  Papaya 1996 (USA, CAN) •  Potato 1998 - 2002

(USA, CAN, MEX, AUS/NZ, JAP, RUS, ROM)

•  Plum 2007 (USA) •  Common Bean 2011 (BRA)

DuPont 1997/2011 (CHN, USA, CAN, MEX,

AUS, NZ, JAP, KOR, TWN)

Monsanto 2011/2012 (USA, CAN, MEX, AUS,

NZ, JAP, KOR, COL)

BASF amylopectin

potato 2010 (EU)

Simplot

Innate™ potato 2014 (USA)

Okanagan

Arctic® Apple 2014 (USA, CAN)

Monsanto

Reduced Lignin Alfalfa

2014-2016 (USA, CAN, MEX,

AUS, NZ, JAP, KOR)

RNAi is not new to agriculture Registered products mainly focus on virus resistance, compositional and quality traits

Monsanto CRW3 for Insect Control Expresses DvSnf7 RNA transcript targeting western corn rootworm

(Diabrotica virgifera) 2014-2016: USA, CAN, MEX, AUS, NZ, TWN, JAP (stage 3), ARG , KOR

8


dsRNA-based products at State Level: Like other biopesticides

• EPA OPP BPPD data reviews and risk assessment •  Biopesticide Registration Action Document

(BRAD) • EPA OPP Proposed registration decision public comment period • EPA OPP BPPD approved label •  Active ingredient content, precautionary

language, and directions for use • State application for registration • For data reviewing states - Biochemical biopesticide dataset

9


Barriers Limit Uptake of Ingested dsRNA

Systemic Barriers q Saliva q Stomach acid q Pancreatic nucleases q Intestinal epithelium q Vascular endothelium q Blood/systemic

nucleases

Cellular Barriers q  Plasma membrane q  Endosomal sequestration q  Lysosomal degradation

9 Monsanto Company

Petrick, J.S. et al., (2013) Reg. Toxicol. Pharm., 66, 167-176


�  RNA in its many forms is a common part of diets from both plant and animal sources

(Ivashuta, et al., 2009; Jensen, et al., 2013; Petrick et al., 2013) �  Multiple studies looking for the uptake of RNA from food have

failed to find significant presence and regulation in mammals (Witwer et al, 2013; Snow et al., 2013; Dickinson et al., 2013; Tosar et al., 2014)

RNAhasafavorableprofileformammalianconsumers

Manybarrierscontributetoprotec0on

X

11


Multiple Interdependent Factors Play a Role in Assessing RNA Ecological Risk

12


®

Pollen Grain

Soil

Early studies show degradation comparable or better than Bt-proteins

�  dsRNA can be accurately measured and evaluated from a variety of sources

�  dsRNA quickly degrades (less than 24 hours in soil, 96 hours in surface water : sediment, ~72 hours in hives)

Hive Armstrongetal.(2013)JAg&FoodChemistry61:12557Dubelmanetal.(2014)PLoSONE9:e93155Fischer,etal.(2016)EnvTox&Chemdoi:10.1002/etc.3585Garbian,etal.(2012)PLoSPath8:e1003035

RNA has a favorable environmental profile

13


Barriers and Differential Taxonomic Responsiveness Limit Potential Invertebrate NTO Hazard Extremely high concentrations of dsRNAs are required to elicit a RNAi phenotype in insect Orders outside Coleoptera1, 2

In some insect Orders barriers to efficacious RNAi-mediated insect control can include: •  Salivary, midgut, and hemolymph

nucleases that rapidly degrade dsRNA3,4

•  Inefficient uptake by midgut epithelial cells

•  Length of dsRNA (e.g. ≥ 60 nts) required for uptake5

14 1.Huvenne, H. and G. Smagghe. 2010.; 2. Baum, J.A. and J. Roberts. 2014.; 3. Allen M.L. and W.B. Walker. 2012; 4. Garbutt, J.S. et al., 2013.; 5. Bolognesi R., et al. 2012.


Velez et al. (2015) Further Supports Lack of Responsiveness of Honey Bees to Environmental dsRNA

Honey bee larval development, adult eclosion, and adult survival were unaffected by D. virgifera and A. mellifera dsRNA targeting v-ATPase (with 21mer alignments)

Exposure at very high levels; 1µg/individual or 1000x the reported LC50 for WCR

Velez A., et al 2015 Chemosphere. 144:1083-90

15


Existing regulatory framework is adequate for dsRNA based products

RNA-based technology has high potential for taxonomic specificity and low likelihood of adverse effects to NTOs

Bioinformatics cannot be used as standalone information for an ecological assessment

There is extremely low potential for off-target effects from saturation of RNAi machinery, immunostimulation and suppression transitivity in NTOs

Key factors that drive potential ecological risk are exposure, responsiveness, and sequence

Ecological testing for RNA-based products can be adequately assessed within the existing framework using properly designed Tier 1 assays to evaluate endpoints relevant to protection goals for NTOs

16 16


Note: Gleit, et al. Prospective Large-Scale Field Study Generates Predictive Model Identifying Major Contributors to Colony Losses

PLOS Pathogens | DOI:10.1371/journal.ppat.1004816 April 13, 2015

•  Currently EPA approved products for Varroa destructor include conventional chemicals and/or essential oils and organic acids

•  Resistance has rendered 2 chemistries ineffective ~~ evidence of resistance to amitraz

17


0

4

8

12

16

0 4 8 12

2014 and 2015 field trial demonstrated significant mite suppression and improvement in hive survival

2015 VARROA CONTROL TESTING– REHOVOT, ISRAEL

At week 11, Varroa mite count reduced by more than 2.1X with BioDirect treatment vs untreated

hives

WEEKS

NON-TREATED

BIODIRECT

# o

f M

ITES

/1

00

BEE

S

Su

rviv

al %

0 1 2 3 4 5 6 7 8 9 10 11 12 30 40 50 60 70 80 90

100

WEEKS M

ites

/ 1

00

bee

s

NON-TREATED

BIODIRECT

2014 VARROA CONTROL TESTING– FL, USA

At week 12, hive survival improved by more than 1.8X with BioDirect treatment vs untreated

hives

18


BioDirect™ for Varroa control does not appear to influence bee survival in the absence of Varroa • Bees were treated with 1X and 10X doses of BioDirectTM and reared in boxes for three weeks • 5 boxes with 30 bees in each treatment group

Bee

su

rviv

al

DAYS

Oligo A

Oligo A

Oligo B Oligo B

19


Flea beetles are highly destructive defoliators of canola causing significant economic yield loss

Attacks young plants resulting in yield loss, estimated at >$300 million damages in North America alone

FleaBeetlesaredevasta>ngpestsofCanola/Rapeseed

Stripedfleabeetle

Cabbagestembeetle

Cruciferfleabeetle

n  Growers in Europe under pressure

n  Potentially provides an alternative solution

20


BioDirecttreatmentsshowefficacyonfleabeetlesincontrolledenvironmentassaysandinthefield

+BioDirect- BioDirect +BioDirect- BioDirect

35Daysa[er2ndspray

Field trials

Negative control BioDirect

Untreated

0

2

4

6

8

10

12

0 5 10 15

% S

urvi

vors

hip

Days Post-Infestation

100

80

60

40

20

Canola leaf disk assay

Negativecontrols

BioDirect 0.2X

BioDirect 1X

Whole plant assays

21


In closing Existing Regulatory Paradigm – including at the state level - for biopesticides is appropriate for dsRNA-based products

Active research •  BioDirect projects are in an early phase of

development

When developed, product concepts will inform problem formulation and regulatory testing plan

Thank you for the opportunity

22

Documents

Topical Application of dsRNA for Pest Management · Flavr Savr tomato Virus resistance Soybean oil composition Quality Traits Calgene 1994/1995 (USA, CAN, MEX) • Squash 1994 (USA,