10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.

1. A perspective on risks associated with
dsRNA-based Products
Prof Neena Mitter
Director, Centre for Horticultural Science,
QAAFI, The University of Queensland, Australia
n.mitter@uq.edu.au

2. Global agriculture
 10 billion people by 2050
 2 trillion calories will be required
per day
 Increased productivity- Reduce crop
losses
 Concerns with current crop
protection measures
 INNOVATION IS KEY DRIVER
http://civileats.com/category/environment/pesticides/
PESTICIDES:
 RESISTANCE
 RESIDUE
 RUN-OFF
 LACK OF
SPECIFICITY
 NEW CHEMICALS

3. RNA interference for crop protection
In transgenic or GM plants pathogen specific
dsRNA is integrated into the genome of the plant to
afford protection
Dzu.doodles.com
• Community acceptance
• Concerns regarding environmental
impact
• Regulation of use
• Cost and time involved
• Lack of transformation protocols

4. Is there another way?
Can we deliver dsRNA as a spray instead of making
a GM plant?
http://www.naturalnews.com/gmos.html

5. dsRNA spray to control virus infection
Pepper Mild
Mottle Virus
dsRNA +
Pepper Mild Mottle Virus
Tenllado et al. (2003)

6. dsRNA-based products act on the RNAi pathway
 Active against virus,
insect and fungal
pathogens
Christiaens et al., 2016
Koch et al., 2016
Wang et al., 2016
Gan et al., 2010

7. dsRNA-based products
 Pre- and post-harvest topical
application of dsRNA-based
products represents a
paradigm shift
 Increased pathogen specificity
 Deployment of new sequences
to address resistance
 Finite amount of dsRNA
https://negativespace.co/plant-grow-hand/

8.  Unstable
 Degradation on plant surface
 Not protected from UV and sunlight
 Can get easily washed off after spray
 Short window of protection
Issues with spray of naked dsRNA

9. How can we convert naked dsRNA
experiments into a commercially
viable system for farmers?
Stabilise?
Stick to the leaf?
Protect from rain?
Non-toxic?
Degradable?
Environmentally
friendly?
Easy to adopt?

10. Formulation
 A formulated dsRNA product
‒ dsRNA carrier
‒ Surfactants
‒ Stabilizers
‒ Buffers
‒ Anti-foam materials
‒ Sticking agents
 Associated risks are on a case-by-case basis
https://pixabay.com/fr/photos/feuille-goutte-d-eau-goutte-macro-1422177/

11. Nanomaterials as carriers of dsRNA
 Clay
 Silica
 Carbon Nanotubes
 Polymers
 Lipid based formulations
 Nanogels
http://www.materials.ox.ac.uk/research/researchareas/nanomaterials.html

12. Clay particles as carriers for non-GM,
delivery of RNA interference
BioClay- Sustainable crop protection
Neena Mitter
Gordon Xu
Max Lu

13. Case study : BioClay
dsRNA loaded on to
layered double
hydroxide
nanoparticles (LDH)
• Increases dsRNA
stability and period
of protection from
days to weeks

14. BioClay- It works
Mitter et al, Nature Plants 2017

15. BioClay version 2
 Specifically designed for targeting insect pests
 pH switch to release dsRNA under alkaline conditions
 Protection from nucleases

16. DNA nanostructures coordinate gene silencing in
mature plants –
 DNA origami or nanoscale folding of DNA
 DNA nanostructures could be loaded with siRNA, to accomplish gene silencing
in plant
 Prior work shows that DNA based nanostructures do not exhibit toxicity in
mammalian systems
 DNA nanostructures do not induce a stress response in plants
Zhang, H….. Landry, MP PNAS (2019)
https://www.beautifulchemistry.net/dna-nanostructures

17. Nanoparticles to deliver genes into plant chloroplasts.
Kwak, SY… Strano, MS Nature Nanotechnology, 2019
Chloroplast Transformation
Lipid exchange envelope
penetration – LEEP
http://news.mit.edu/2017/engineers-create-
nanobionic-plants-that-glow-1213

18. Nanomaterials as carriers of dsRNA
 Releasing active ingredients in a slow/targeted manner
 Protecting the active ingredient against premature degradation
 The context within which a material is being evaluated
 The importance of measuring a specific parameter within that
context
 The feasibility of measuring the parameter within a specific context.
Oberdörster et al (2005)
APVMA report 2015, www.apvma.gov.au.

19. Putative risks of dsRNA-based products
Human health
Environment
Trade

20.  Human exposure pathways:
‒ Ingestion
‒ Inhalation
‒ Dermal
 Environmental exposure:
‒ Mammals
‒ Fish
‒ Fungi
‒ Soil biota etc.
 Trade impacts:
‒ Non-GM designation
‒ Withholding periods
Putative risks of dsRNA-based products
https://www.greatersudbury.ca/live/environment-and-sustainability1/
https://www.pexels.com/photo/aerial-view-of-cargo-ship-1554646/ https://www.corporatebenefitsauthority.com/

21. Risks to human health – oral exposure
 Multiple and redundant biological
barriers to absorption and/or
biodistribution
 dsRNAs and siRNAs consumed in
significant quantities via a diet of
fruit and vegetables without
detectable effect
 miRNA studies indicate insufficient
uptake for biologically relevant
effects (e.g. Sherman et al. 2015;
Snow et al., 2013) https://www.pexels.com/photo/flat-lay-photography-of-vegetable-salad-on-plate-1640777/

22. Petrick et al., 2015 – dsRNA and siRNA 28-day
mouse feeding trials
‒Treatments included 218 nt dsRNA and 4 x 21 nt
siRNAs with 100% homology to mouse vATPase
transcript
‒No evidence of differential gene expression
relative to controls
‒No evidence of impacts on body weight, food
consumption, clinical observations, clinical
chemistry, hematology, gross pathology, or
histopathology endpoints
Risks to human health – oral exposure
https://www.pexels.com/photo/grey-mouse-carrying-food-736524/

23. Food Standards Australia New Zealand on dsRNA
Consumption
Investigation of the safety of consuming dsRNAs generated via GM
crops (2013)
1. Gene silencing or RNAi is a universal mechanism that is naturally
present in eukaryotic organisms
2. dsRNAs are a normal constituent of the human diet
3. A number of barriers exist to the systemic and cellular uptake of
exogenous nucleic acids, including small RNAs, by humans
4. There is no scientific basis for presuming that dsRNAs produced
by GM plants would pose a greater risk than dsRNAs naturally
present in food
http://www.foodstandards.gov.au/consumer/gmfood/Pages/Response-to-Heinemann-et-
al-on-the-regulation-of-GM-crops-and-foods-developed-using-gene-silencing.aspx

24.  Dermal and inhalation exposure
‒ Little evidence of transmembrane movement without
conjugation or encapsulation
 Low likelihood of allergenicity as no new protein
produced
Risks to human health – inhalation and dermal
exposure

25. Risks to the environment – Off targets
Species closely related to the
target pathogen most likely to be
affected
‒Similar uptake mechanism
‒Target transcript homology
Key question: what level of
dsRNA homology to a non-target
sequence is sufficient to induce
biological effects? https://www.mhtpmedicalgenomics.org.au/index.php/services/bioinformatic
s

26.  Non-formulated dsRNA rapidly degrades in the environment,
presenting few risks related to persistence
Risks to the environment - persistence
 No basis for
concentration of
topically-applied dsRNA
in higher organisms in
the food chain
https://sydney.edu.au/news-opinion/news/2016/08/29/innovation-boosts-wheat-production-in-india.html

27. Risks to trade – GMO declaration
Natural
virus
infection
Topically applied dsRNA
• No promotors or
terminator regulatory
sequences
• No transgene insertion
• Not heritable / cannot
cross pollinate
• No protein expression

28. Risks to trade – GMO declaration
 In Australia, the Office of the Gene Technology Regulator has
proposed that topically-applied dsRNA be exempt from GMO
regulations (Schedule 1A – Techniques that are not gene technology)
Presentation at the CSIRO Gene Editing of Crops Workshop
28-30 November 2017

29. Naked dsRNA:
‒Short or nil withholding periods (set to allow chemical
residues in edible commodities for domestic markets)
‒Short or nil export intervals (to satisfy the standards
imposed by overseas trading partners)
Trade – withholding periods

30. DsRNA applications for the ‘Agriculture of Tomorrow”
Design of regulation
and public opinion
are crucial
FUNGI
Hailing Jin, Nature Plants , 2016
VIRUSES INSECTS PROTECTED
CROPPING
ANIMAL HEALTH
BIOSECURITYPACKHOUSES
Endless possibilities……