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. RNAi as a novel technology in
pest control: current status and
challenges
Olivier Christiaens

2. Introduction to RNAi
What is RNA Interference (RNAi)?
• Post-transcriptional gene
silencing
Gene silencing
• mRNA degradation to prevent
formation of protein

3. Cellular functions:
• Protection against viruses (siRNA)
• Protection against ‘jumping genes’ (piRNA)
• Internal gene regulation (miRNA)

4. RNAi as a pest control technology
Joga et al., 2016; Frontiers in Physiology

5. dsRNA
Extracellular
Cytoplasm
Dicer
Dicer
Ago
RISC
RISC RISC
mRNA
siRNA
mRNA degradation
Passenger
strand
Guide
strand
Ago Ago
Molecular mechanism of the siRNA pathway

6. RNAi as a crop protection technology
First proof of concept: Baum et al., 2007; Nature Biotechnology
After Kupferschmidt, 2013; Science

7. Gut lumen
Haemolymph
RNAi as a crop protection technology
Baum et al (2007), Nature Biotechnology 25

8. RNAi as a crop protection technology
Protection of beneficial insects against viruses and parasites
Deformed wing virus
(DWV)
DWV-dsRNA
 DWV-dsRNA is mixed with food
 Virus infection
• Desai et al 2012 (Insect Mol Biol vol 21)
• Varroa destructor control (Monsanto/Bayer)

9. Efficiency of RNAi in arthropods:
• Very variable
Coleoptera > Diptera, Hemiptera > Lepidoptera
Factors affecting RNAi sensitivity
• Degradation of dsRNA (saliva, gut, haemolymph)
• Uptake of dsRNA in cells (Sid-like channels and/or endocytosis)
• Endosomal release in the cell
• Spreading and amplification of RNAi signal (no RdRP in insects)
• Virus infections (saturation or VSPs)
RNAi as a crop protection technology

10. Not all Coleoptera are highly sensitive…
RNAi efficiency
Cylas (African sweet potato weevil)
• Much lower sensitivity upon ingestion
of high amounts of dsRNA
• Very high sensitivity with injection
• High silencing efficiency
• Silencing lasts up to 10 days after
single injection
Adult-
control
(dsGFP)
Adult (dsLac2)
Prentice et al., 2015

11. RNAi efficiency
Oral delivery of dsRNA in Cylas
C. puncticollis C. brunneus
C C 3h 5’ 15’ 30’ 1h 2h 3h C C 3h 5’ 15’ 30’ 1h 2h 3h
5’ 15’ 30’ 1h
+ 20mM EDTA
5’ 15’ 30’ 1h 3h 16h
L. decemlineata
Prentice et al., 2016, Pest Management Science
Christiaens et al., 2016, Scientific Reports

12. RNAi-based insect pest control
- Host-induced gene silencing (HIGS)
- Spray-induced gene silencing (SIGS)
- Virus-induced gene silencing (VIGS)
- Trunk injections
- Root absorption
Depends on insect pest, regulations, efficiency, formulations
DsRNA delivery:

13. dsRNA delivery
• Host-induced gene silencing
Constant dsRNA exposure, long term control
No spraying necessary
dsRNA mostly diced to siRNA by plant siRNA machinery
Regulation, cost, public acceptance

14. • Virus-induced gene silencing
 Plant virus delivery
 Insect virus delivery
dsRNA delivery
Insect cell
Host genome
Target gene
transcription
Target mRNA
Replication
Gene silencing
Engineered virus
expressing insect
target gene
fragments
Virus genome
Nucleus

15. • Non-transgenic in planta delivery
dsRNA delivery
Trunk injection Root absorption

16. • Topical delivery (spraying)
 Naked dsRNA
Natural molecule, short environmental fate, biopesticide
RNAi efficiency, short environmental fate
 Formulations/carriers
Increased persistence, cellular uptake  efficiency
Potential implications for biosafety?
dsRNA delivery
 Bacterial systems
Increased persistence and efficiency
Regulation as GMO?

17. • Formulations
- Increased cellular uptake
- Protection from (nucleolytic) degradation
 Lipid-based encapsulation
 Lipofectamine (Whyard et al., 2009)
 Structural changes to the dsRNA
 Increase persistence and/or cellular uptake
 Examples:
- Trillium Ag
- NanoSur
Topical dsRNA delivery
www.AgroSpheres.com
 Bacterial minicells (AgroSpheres AgriCell)

18. • Formulations
 Bacterial systems
Topical dsRNA delivery
Whitten et al., 2017; BioEssays

19.  Sustained release on plants
Topical dsRNA delivery
- Example: Bioclay (Mitter et al., 2017; Nature Plants)
@NaturePlants

20.  Cell penetrating peptides
CPP dsRBD
Topical dsRNA delivery

21.  Viral-like particles
Topical dsRNA delivery
 Polymers

22.  Polymers
• Spodoptera exigua
• Low sensitivity for RNAi
• Rapid nucleolytic degradation of dsRNA
in the digestive system
• Very alkaline gut lumen
Topical dsRNA delivery

23. Topical dsRNA delivery
 Polymers

24. dsRNA +
polymer
dsRNA
• CY3-labeled
dsRNA
• FITC-labeled
polymer
• CF203 cells
• 10’ incubation

25. Environmental safety
• RNAi-mediated crop protection
 Biopesticide (?)
 Molecule ubiquitously present in nature
 Sequence-dependent mode of action (potential for selectivity)
 Short persistence in the environment
However…adverse effects on non-target (insect) species are
possible
 Sequence-dependent effects  dsRNA design is critical
 Sequence-independent effects

26. Literature review of baseline information to support the risk assessment of RNAi-derived GM plants
 Cellular uptake and systemic spread of sRNAs
 Plausible routes of exposure
 RNAi efficiency and factors that influence this
 Plausibility of unintended effects
 Overview of available genomic data in invertebrate species and a
discussion on the potential role of bioinformatics in RNAi ERA

27. Thank you for your attention!
Dr. ir. Olivier Christiaens
Department of Plants and Crops
Ghent University – Belgium
olchrist.christiaens@ugent.be