Presented by
Raquel Adams & Richi Gupta
Binf 705- Research Ethics
 Background
and History of RNAi
 What is RNAi?
•
•
•
•
Molecular mechanism
siRNA vs miRNA
Applications of RNAi
Limitations of RNAi
 RNAi
Beneficial or Harmful?
 Potential concerns
 EPA Laboratory Assay
 References


In 1990,Nepoli and Jorgenson were first to report
RNAi type phenomenon in Petunias. Named this
phenomenon as “co-suppression”
In 1990’s, other scientists also observed
independently that RNA inhibited protein expression
in plants and fungi.
• Plants - post-transcriptional gene silencing (PTGS)
• Fungi - quelling

In 1998, Andrew Fire and Craig Mello discovered the
phenomenon of RNA interference or RNAi. They
awarded the 2006 Nobel price for Physiology or
Medicine for their discovery.




Set of related processes in which
small regulatory RNAs direct
sequence-specific repression of
gene expression
Evolutionary ancient and various
versions are found in eukaryotic
organisms.
Some are induced by invasive
nucleic acids (e.g. viruses,
transposons, or transgenes, and
serves to defend the host plant
against such invaders) – short
interfering RNA (siRNA) pathway.
Some are endogenous pathways
used to control an organism’s own
gene expression- micro RNA
(miRNA) pathway

siRNA based approach
• Hairpin transgenes or RNAi construct is designed to
produce a population of dsRNA with complementarity to a
chosen target messenger
• Very effective at silencing the target gene as there are
many different small RNAs all targeting the same
transcript

miRNA based approach
• Endogenous miRNA gene is genetically engineered to
produce a transgene in which the endogenous miRNA in
the precursor is replaced with one that is complementary
to the targeted messenger RNA
• Precise, more selective as only one small RNA is
produced rather than a population

Examples of transgenic RNAi trait development
•
•
•
•
•
•
•
•
•
•
Modified oil composition in soybean
Increased lysine content in corn
Reduced caffeine content in coffee
Potato starch composition (e.g., Amflora)
Reduced lignin alfalfa
Reduced phytate sorghum
Delayed softening in tomatoes
Virus resistant plums, papaya, potatoes, squash, etc
Nematode resistant soybean
Target pest that feed on root-knot nematodes, corn
rootworm, and bollworm
 Development of resistance
 Sequence heterogeneity of
siRNA can cause
off-target effects which can be overcome by
miRNA approach
 siRNA-based RNAi strategies might not be
suitable for some applications requiring
tissue-specific silencing of genes
 miRNA-based RNAi gene silencing might
not be very durable because only a single
21- or 22-nt specificity determinant is
involved.
 40
percent of food in the US today goes
uneaten
 Reducing food losses by just 15 percent
would be enough food to feed more than
25 million Americans every year
 Benefits?
• How RNAi Pesticides and Herbicides work?
• The case of Resistance
 Potential
Hazards
 EPA Laboratory Assessments



Could let farmers
alter crops without
permanent genetic
modification.
Ie: spray a crop
with a drought
remedy only when
there’s a drought,
control mosquitoes
in an
environmentally
benign way
Could save the
honey bees?
(neonicotinoid)
 Today, Monsanto
has revenues of about
$9 billion a year from GM seeds for crops
that produce the insect toxin Bt or resist
the weed killer Roundup. GM corn, soy,
and cotton plants now spread across 180
million hectares.
 http://www.technologyreview.com/featur
edstory/540136/the-next-great-gmodebate/
http://www.nytimes.com/2014/01/28/business/energy-environment/geneticweapon-against-insects-raises-hope-and-fear-in-farming.html?_r=0



Monsanto claims that insects won't
likely develop resistance to the RNAi
treatments, as they have to most
chemical treatments in the past.
"This is surprisingly reminiscent
of Monsanto's assurancesin the '90s
that weeds would be very unlikely to
develop resistance to the glyphosate
[Roundup] herbicide…and now we
have an epidemic of glyphosate
resistant weeds.”
http://www.motherjones.com/tomphilpott/2015/08/coming-farm-fieldnear-you-gene-silencing-pesticidesRNA-RNAi
BioDirect Insect Control: Colorado Potato Beetle (Phase II)
BioDirect Bee Health: Aims to control Varroa mites and multiple viruses
(Phase I)
BioDirect Virus Control: Tospovirus Control –against the tomato spotted
wilt virus. (Phase I)
BioDirect Weed Control: Palmer Amaranth + Waterhemp (Phase I)
Usually takes 9-10 years from Phase I of R&D.

degrades quickly in soil

genetically precise enough to kill potato bugs but
spare their ladybug cousins.


So far, consuming RNA molecules appears no more
toxic to people than drinking a glass of orange juice.
As Monsanto put it in a letter to U.S. regulators,
“humans have been eating RNA as long as we have
been eating.”
http://www.technologyreview.com/featuredstory/54
0136/the-next-great-gmo-debate/
immune stimulation,
 saturation of the RNAi machinery
 off-target gene silencing,
 silencing the target gene in unintended
organisms,

 Most
of the cases will be presented via
journals published.
It was found that the injection of small fragments
(fewer than 30 nt) of RNA could stimulate an immune
reaction in mammals (Robbins et al. 2009)
 In mammals, the immunostimulation by RNAi led to
reduced lymphocytes and platelet cells, largely
correlated with cytokine response to the siRNA
(Judge et al. 2005).
 Although there are some similarities in the innate
immune response of insects and mammals, it is
unclear how the immune systems of other organisms
will react to an influx of small RNAs. Nor is it known
how this immunostimulation will affect the fitness of
nontarget organisms (Lundgren and Jurat-Fuentes
2012).

Activation of the mammalian immune system by
siRNAs
Joao T Marques1 & Bryan R G Williams1
Most mammalian cells possess
intracellular pathways that
recognize dsRNA through
cytoplasmic receptors, such as PKR,
OAS and RIG-I. These pathways can
also recognize siRNAs and activate
IRF-3, NF-kappaB and MAPK
pathways leading to the expression
of IFN and pro-inflammatory
cytokines. In addition, activation of
PKR and OAS pathways results in
general protein synthesis inhibition.
In the cytoplasm the siRNAs also
enter the RNAi pathway through
RISC. RISC, RNA-induced silencing
complex; IFN, interferon; PKR,
dsRNA-dependent protein kinase;
IRF, interferon regulatory factor;
RIG-I, retinoic acid inducible
protein; OAS, 2'5'-oligoadenylate
synthetase.
http://www.nature.com/nbt/journal/v23/n11/fig_tab/nbt1161_F2.html
 One in silico examination of sequence homologies between
siRNA sequences and three transcriptomes from diverse
organisms revealed that off- target effects were observed in as
few as 5% and up to 80% of the siRNAs assessed (Qiu et al.
2005).
 17% of siRNAs had complete sequence homologies with offtarget binding sites in the Drosophila melanogaster genome
(Kulkarni et al. 2006).
 These studies indicate that off-target effects of siRNAs used in
RNAi are probably more common than was initially believed;
these effects could have implications for nontarget effects of
GM crops if off-target gene suppression occurs in nontarget
organisms and if these organisms are exposed to RNAi to a
sufficient degree.
 High
levels of exogenous siRNAs can
saturate a cell’s RNAi machinery and
thereby reduce the efficiency at which a cell
regulates endogenous gene expression
(Agrawal et al. 2003, Dillin 2003).
 There is a limited number of RISCs (RNAinducedd silencing complex) present within
a cell, and if the augmented siRNAs saturate
these complexes, the health and
performance of the cell may be
compromised (Kahn et al. 2009).



In developing GM maize plants resistant to
D. virgifera (spotted cucumber beetle).
Baum and colleagues (2007) also
examined the effects of a few of the
dsRNAs identified for plant transformation
on several other beetle species.
dsRNAs that targeted D. virgifera (spotted
cucumber beetle) also reduced survival of
Diabrotica undecimpunctata (western corn
rootworm) and Leptinotarsa decemlineata
(Colorado potato beetle) significantly,
even though these pests shared only 79%
and 83% of their genome, respectively.
A
recent study showed that plant-produced
microRNAs constitute 5%–10% of human
microRNAs and that these are likely taken in
with food (Zhang et al. 2012).
 Their work indicate that “ microRNAs taken
into an animal through food may occur more
often than is commonly thought and may
influence gene expression in nontarget
organisms that ingest siRNAs within plant
tissues.”
 Dr. Vicki Vance, a
biology professor at the
University of South Carolina, found that
dietary RNAs from plants could cross the
gastrointestinal barrier and act as a
potential cancer treatment.
 She publicly cautions on the use of RNAi as
pesticides and Monsanto cancelled her
scheduled address to an international
symposium on the biosafety of GMO food
products.
 She refuses to ignore her findings




1: Safe consumption of RNA - no convincing evidence in
the literature that ingested dsRNA is absorbed from the
mammalian gut in a form that produces biological effects.
2: RNA degradation in the mammalian digestive system
- In the digestive tract and elsewhere, RNA is subject to both
non-enzymatic and enzymatic degradation
3: Barriers to uptake and systemic distribution of
dietary RNA in mammals vs. other organisms Mechanisms for exogenous dsRNA import into RNAi
pathways are not known in mammals, and reports of carrierunmediated small RNA uptake await confirmation
4: Immune stimulation - that there is no clear evidence
that small RNAs could trigger the immune systems of nonmammalian non-target species or what dose is necessary to
trigger a response.
Address nontarget species by administering a
maximum-hazard dose (1–20 times the dose) of the
known environmental exposure concentration.
 Focus on six to eight indicator species:
 honeybees,
 springtails,
 earthworms,
 daphnia,
 predatory beetles or pirate bugs,
 and parasitoid wasps
 They must represent different functional guilds (e.g.,
pollinators, predators, parasitoids, detriti- vores).

PIPs – Plant-Incorporated Protectants
"Big 6" pesticide and GMO corporations
are BASF, Bayer, Dupont, Dow Chemical
Company, Monsanto, and Syngenta.
Companies
working on
RNAi sprays:
• Monsanto
• Syngenta
recently
acquired all
of Devgen for
around $500
million.
• Bayer is also
pursuing the
research
British writer George Monbiot:
“The world has a surplus of food, but still
people go hungry. They go hungry
because they cannot afford to buy it. They
cannot afford to buy it because the
sources of wealth and the means of
production have been captured and in
some cases monopolized by landowners
and corporations. The purpose of the
biotech industry is to capture and
monopolize the sources of wealth and the
means of production …





Agrawal N, Dasaradhi PVN, Mohmmed A, Malhotra P, Bhatnagar RK,
Mukherjee SK. 2003. RNA interference: Biology, mechanism, and
applications. Microbiology and Molecular Biology Reviews 67:
657–685.
Davidson BL, McCray PB. 2011. Current prospects for RNA
interference– based therapies. Nature Reviews Genetics 12: 329–
340.
Dillin A. 2003. The specifics of small interfering RNA specificity.
Proceedings of the National Academy of Sciences 100: 6289–6291.
Jackson AL, Linsley PS. 2010. Recognizing and avoiding siRNA offtarget effects for target identification and therapeutic application.
Nature Reviews Drug Discovery 9: 57–67.
Judge AD, Sood V, Shaw JR, Fang D, McClintock K, MacLachlan I.
2005. Sequence-dependent stimulation of the mammalian innate
immune response by synthetic siRNA. Nature Biotechnology 23:
457–462.






Kahn AA, Betel D, Miller ML, Sander C, Leslie CS, Marks DS. 2009. Transfection of
small RNAs globally perturbs gene regulation by endog- enous microRNAs. Nature
Biotechnology 27: 549–555.
Lundgren JG, Duan JJ. 2003. RNAi-Based Insecticidal Crops: Potential Effects on
Nontarget Species. BioScience 8:657-665.
Lundgren JG, Jurat-Fuentes JL. 2012. Physiology and ecology of host defense against
microbial invaders. Pages 461–480 in Vega FE, Kaya HK, eds. Insect Pathology, 2nd
ed. Academic Press.
Qiu S, Adema CM, Lane T. 2005. A computational study of off-target effects of RNA
interference. Nucleic Acids Research 33: 1834–1847.
Robbins M, Judge A, MacLachlan I. 2009. siRNA and innate immunity.
Oligonucleotides 19: 89–102.
Zhang L, et al. 2012. Exogenous plant MIR168a specifically targets mam- malian
LDLRAP1: Evidence of cross-kingdom regulation by microRNA. Cell Research 22:
107–126.
FIFRA Scientific Advisory Panel Meeting Held at
One Potomac Yard Arlington, Virginia . January 28,
2014. A Set of Scientific Issues Being Considered by
the Environmental Protection Agency Regarding:
RNAi Technology: Program Formulation for Human
Health and Ecological Risk Assessment. SAP Minutes
No. 2014-02.
 http://www.technologyreview.com/featuredstory/54
0136/the-next-great-gmo-debate/


http://www.motherjones.com/tomphilpott/2015/08/coming-farm-field-near-you-genesilencing-pesticides-RNA-RNAi