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. Dietary uptake of environmental
dsRNA in humans and other
vertebrates
Thaís Rodrigues
trodrigues@greenlightbio.com

2. 2
Presentation Outline
RNAi in vertebrates
Mechanism of RNAi (external dsRNA/siRNA)
History of safe consumption
Absence of efficacious oral RNAi therapeutics
Biological barriers
Published Studies
Mammals
Non-mammalian vertebrates
Human Health Considerations
Summary and Take home message

3. 3
RNAi mechanism
Cytoplasm
siRNAdsRNA
Dicer
RISC
RISC assembly
RISC activation
Target
recognition
Target mRNA
cleavage Target mRNA
Antisense strand
Sense strand
cleavage
Dose-dependent:
No amplification system
1Koenig et al., (2013) Pharmaceuticals 6:881-914
Sequence-dependent
siRNA duplex

4. 4
History of Safe Consumption
Abundance of endogenous RNAs with 100% matches to key
transcripts of humans and other animals are safely consumed
Staple crops: conventional corn, soybean, and rice 1,2
Fresh market vegetables: tomato, melon, zucchini, and onion 3
RNAi is not new to agriculture and underlies many domesticated
crop phenotypes, as well as approved biotech crop traits 4
1Ivashuta et al., (2009) Food Chem. Tox. 47, 353-360
2Frizzi et al., (2014) J. Ag. Food Chem. 62, 12067-12074
3Petrick et al., (2016) Reg. Toxicol. Pharm. 81, 57-68
4Sherman et al., (2015) Reg. Toxicol. Pharm. 73,671-680

5. 5
Oral delivery is the most desirable route for drugs but it is not
viable to date
Without enhancers, oral bioavailability of dsRNA to mammal cells is
≤ 1%1
RNA drugs are extensively metabolized after injection, half-
life of ~5 mins 1,2,3
Absence of efficacious oral RNAi therapeutics
WHY?
1Reviewed by Petrick et al., (2013) Reg. Toxicol. Pharm., 66, 167-176
2Christensen et al., (2013) Drug Metab. Distr. 412, 1211-1219
3Thompsonet al., (2012) Nucleic Acid Ther. 22, 255-264

6. 6GreenLight Biosciences, Inc. — Confidential
Initial dsRNA degradation starts before ingestion
Degradation is ongoing during harvest, transportation, and
storage
Highly processed foods and food preparation further accelerate
RNA degradation
For all food sources, mechanical, chemical and enzymatic
processes expose RNAs during digestion

7. 7
Dicer
cleavage
RISC
assembly
mRNA
cleavage
1
2
3
Cellular Barriers
Achieve target cell
Endocytosis
Endosome escape
Gene function
2
3
4
1
4
Cellular
Achieve target cell
Endocytosis
Endosome escape
Gene function
2
3
4
1
dsRNA Dicer
cleavage
RISC
assembly
mRNA
cleavage
1
2
3
4
1
2
3
Bloodstream
Serum Nucleases
Renal filtration (Kidney)
Vascular endothelium
2
3
1
1
2
3
Reviewed by 1Artiga et al., (2019) J. Mater. Chem. B. 7, 876; 2Sherman et al., (2015) Reg. Tox. Pharm. 73, 671-680;
3FIFRA SAP Minute No. 2016-02 (2016); 4Petrick et al., (2013) Reg. Tox. Pharm. 66, 167-176
2
Gastrointestinal
Saliva
Stomach acids/digestive enzymes
Pancreatic nucleases
1
3
2
Biological Barriers

8. Series of events would have to occur in order for an exogenous
dsRNA to harm a vertebrate
8
Expose
Import into the cell cytoplasm intact
Unlikely at dietary uptake level
Deliver to target tissues
Gene silencing results in harmful effects
Escape biological barriers
Image from Petrick et al., (2013) Reg. Tox. Pharm. 66, 167-176

9. 9
Ingestion of chemically synthesized RNA molecules has
proven to be an exceedingly challenging route for effective
uptake and biological activity 1
Inherent instability of naked RNA oligonucleotides
Harsh environment of the gastrointestinal tract
Lack of molecular transporter in the gut for access to the systemic
circulation
Zhang et al. (2012) reported from dietary sources:
1) Plant miRNAs were transferred into blood and tissues
2) Regulated a mammalian transcript involved in cholesterol
homeostasis (LDL) 2
1Reviewed by Sherman et al., (2015) Reg. Toxicol. Pharm. 73,671-680
2Zhang et al., (2012) Cell Res. 22, 107-126
Published Studies

10. 10
Published Studies in mammalian
Snow et al. (2013) studied a) one conserved mammalian miRNA
(miR-21) and b) three conserved plant-derived miRNAs
1Snow et al., (2013) RNA Biol. 10,1107-1116
Mouse genetically null for
miR-21
a) Animal fat (miR-21)
negligible expression
plasm or organ tissue
b) plant-derived miRNAs
no detectable levels
human plasma
insignificant levels

11. 11
Published Studies in mammalian
Witwer et al. (2013) studied miRNA content of macaques after
oral ingestion of a plant miRNA-enriched shake
low level and no correlation with plant-miRNA ingestion1
Dickinson et al. (2013) – replicate of Zhang et al. (2012)
Rice-derived miRNAs detected in mouse plasma did not correlate
with levels of dietary consumption
Fasting and an unbalanced nutritional intake drove the alterations in
LDL originally reported by Zhang et al. (not diet-derived miRNAs) 2
1Witwer et al., (2013) RNA Biol. 10, 1080-1086
2Dickinson et al., (2013) Nat. Biotechnol. 31, 965-967

12. 12
Published Studies in non-mammalian vertebrates
No successful responses by
feeding with naked dsRNA 1,2
Transfection agents showed
RNAi response is sequence-
specific 1,3,4
Organism Species
South Africa clawed frog Xenopus laevis
Zebrafish Dania rerio
Rainbow trout Onchorynchus mykiss
Carp Cyprinus carpio
Chicken Gallus gallus
Red eared slider turtle Trachemys scripta
Sea Lamprey Petromyzon marinus
Songbird Zonotrichia leucophrys gambelii
Bobwhite quail Colinus virginianus
1Reviewed by Sifuentes-Romero et al., (2011) Mutat Res Rev Mutat Res. 728, 158-171
2Bachma et al., (2016) Regul Toxicol Pharmacol. 81, 77-88
3Ukuba et al., (2012) PLoS ONE 7(1): e30202
4Heath et al., (2014) PLoS ONE 9(2): e88387

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Human Health Considerations
Passage of large and hydrophilic molecules (nucleic acids
>10kD) through skin is reduced to virtually negligible levels
without the addition of a transport enhancement strategy (such as
microneedles and nanoparticles) 1
The combination of RNases and acids found in the human digestive
system are likely to ensure that all forms of RNA structure are
degraded throughout the digestive process 2
Non-specific effects, such as innate immune system stimulation
or saturation of native RNAi machinery, is highly unlikely to happen
because dietary RNA is ingested 3 1Ivashuta et al., (2009) Food Chem. Tox. 47, 353-360
2Reviewed at FIFRA SAP Minute No. 2014-02 (2014)
3Reviewed at FIFRA SAP Minute No. 2016-02 (2016)

14. 14
Summary
RNAi in vertebrates
Mechanism of RNAi is dose-dependent and sequence-specific
RNAs has been safely consumed in fruits, vegetables, and crops
Although highly desired, to date, there are no successful oral RNAi drugs
Initial dsRNA degradation starts before ingestion
Simultaneous barriers at gastrointestinal, bloodstream and cellular levels
make RNAi responses from oral exposure unlikely
Negligible levels and non-correlated to dietary exposure

15. 15
Take home message
SYSTEMIC ABSORPTION AND BIOLOGICAL EFFECTS
by dietary exposure in mammals and other vertebrates of
Naked molecules
HIGHLY IMPROBABLE
Transfection agents
SEQUENCE/DOSE
DEPENDENT

16. Thaís Rodrigues
trodrigues@greenlightbio.com