Limits of Concern for the Risk Assessment of GMP · 2017-09-18 · „Limits of Concern for the risk assessment of GMP” 3 LoCs and protection goals Role of LoCs in the stepwise

1

EFSA, Parma 24th May 2017 Marion Dolezel

Limits of Concern for the Risk Assessment of GMP

„Limits of Concern for the risk assessment of GMP”

2

Federal Agency for Nature Conservation, Unit II 3.3. (Risk

Assessment GMO)

UFOPLAN 2013 (testing & development project)

October 2013 – June 2017

Aims of the project:

evaluation

operationalisation

exemplification


3

LoCs and protection goals

Role of LoCs in the stepwise testing approach

Relationship between the comparative safety assessment

and LoCs

LoCs and long-term effects

LoCs for certain areas of risk

LoC values for non-target organisms practicable and

reasonable

LoCs for species of conservation concern


4

Literature / background information

Stakeholders interviews

Topic-related workshops with scientific experts

Stakeholder-Workshop

2 scientific publications Dolezel et al. (2017). Are Limits of Concern a useful concept to

improve the environmental risk assessment of GM plants? Environ Sci Eur (2017) 29:7; doi 10.1186/s12302-017-0104-2

Final report

public presentation

Background

5

Background

6

„…the level of environmental protection to be preserved is expressed through the setting of limits of concern…“

Background

7

natural resources or natural resource services

EU legislation

thresholds for acceptable adverse effect(s) for ERA purposes

Background

8

ERA of applicants:

stat. sign. difference ≠ biological relevance

qualitative risk characterisation: negligible/not likely

EFSA objectives:

stat. sign. differences = biological relevance ?

quantitative risk characterisation: effect size = LoC ?

Definition LoC (EFSA 2010)

9

“…the minimum ecological effects that are

deemed biologically relevant and that

are deemed of sufficient magnitude to

cause harm”

LoCs and stepwise testing strategy (EFSA 2010)

10

Operationalization of the LoC concept (EFSA 2010)

11

protection goals relevant for the ERA

protection goals in the EU

assessment endpoints

measurement endpoints

Limit of Concern

Abundance? Mortality? Weight? …

Open questions & challenges I

12

Lack of harm definition for PGs

In ERA: damage to PGs cannot be tested directly – use of

proxies

Ecological entities assessed ≠ protected entities

Spatial & temporal scales

Lack of scientific knowledge on ecological significance of

adverse effects

consequence for definition of LoC

13

Integration of LoCs into current ERA system

14

Open questions & challenges II

What shall a LoC constitute (trigger value, stop criterion)?

What is the consequence of an exceedance/non-

exceedance?

15

LoC in the stepwise testing approach

16

Open questions & challenges III

Stat. sign. differences or non-equivalences need to be

followed up (EFSA 2010)

Assessment of their toxicological or biological relevance

taking safety limits into account (EFSA 2010)

Results of comparative assessment may be relevant for LoC

concept

But usually not done in ERA practice

Example GM soybean & lectins – effects on NTOs

17

LoCs and comparative safety assessment

18

Open questions & challenges IV

LoCs should be derived from

EU-wide protection goals

and should be valid for all

receiving environments

19

LoCs and receiving environments

20

Open questions & challenges V

Define LoCs if long-term effects are likely to occur and if

risk hypothesis can be formulated

Requires risk management measures and post-market

environmental monitoring

21

LoC Definition

Acceptability threshold (quantitatively, qualitatively) for

adverse effects on entities, functions, processes …

…that trigger regulatory concern …

… that have the possibility to cause harm to the relevant protection

goal(s) (EFSA 2010)

… or because these adverse effects are valued as being important for a

specific protection goal (see EFSA SC 2011)

22

Operationalisation of LoCs

3 Examples (risk areas):

Outcrossing, persistence, invasiveness (HT oilseed rape)

Impacts on NTOs (Bt maize)

Effects due to changes in cultivation & management

methods (HT crops)

23

Operationalisation of LoCs

3 Examples (risk areas):

Specific protection goals

Indicators to assess effects (based on Kowarik et al. 2008)

Existing thresholds

Aspects to consider for setting LoC

Dolezel et al. (20xx): in prep.

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Definition of LoCs - Example Bt maize

effect/risk

effect/risk

GM pollen

possible starting point for LoCs

reduction in population size of faunal species

toxic effects on test species

exposure

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Example: Bt Maize – NT Lepidoptera

biodiversity and agricultural protection goals

Proposals for LoCs

LoCs for lab studies: validation of thresholds needed

LoCs for field studies: effects on less mobile larval stages

exposure-based LoCs: amount of pollen deposited on field margins

differentiation of LoCs between in-crop and off-crop (see PPPs)

EFSA opinions (2011, 2012, 2015, 2016)

Ecosystem service concept for ERA (EFSA SC 2016)

negligible effects for NT Lepidoptera


• ERA of Bt maize (MON810, Bt11, 1507)

Source Protection object Protection levels

EFSA (2011, 2012) NT lepi in within maize fields & margins < 1 % global larval mortality

EFSA (2011, 2012) NT lepi of conservation concern in protected habitats

< 0,5 % local larval mortality

EFSA (2015) NT lepi of conservation concern in protected habitats

< 0,5-1 % larval mortality

EFSA SC (2016) Lepi as service providing unit for ES < 1 % reduction in abundance < 1 % global mortality

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27


• dose-response relationships for Lepi larvae and Bt maize pollen (e.g. Holst et al. 2013, EFSA 2011)

• realistic pollen deposition data on food plants (e.g. Hofmann et al. 2016)

28

Conclusions

LoC – useful concept for ERA of GMOs

Further elaboration and specifications

LoC = scientific & political decision

Potential overlap with ES concept

Lack of scientific knowledge should not prevent defining LoCs

Enables harmonization between ERAs of different stressors

Decisions on exceedances of LoC before concept is applied

Transparency

Increased confidence in conclusions of risk

29

Marion Dolezel

Landuse & Biosafety

Environment Agency Austria

Tel.: +43-(0)1-31304 3120

Email: [email protected]

www.umweltbundesamt.at

http://www.umweltbundesamt.at/

dsRNA & miRNA pathways Baseline information to support the risk assessment of RNAi-based GM plants

Petr Svoboda Institute of Molecular Genetics AS CR, Prague

responses to double-stranded RNA

A U C G A U C G A U C G

A U C G A U C G A U

RNA replication

RNA basepairing

Complementarity in nucleic acids

A U C G A U C G A U C G

A U C G A U C G

Sources & types of dsRNA

Exogenous

viral life cycle

inverted repeats convergent transcription pairing in trans

Endogenous

• frequently associated with parasitic mobile elements

genome

dsRNA response

adenosine deamination

interferon response

innate immunity

SEQUENCE-INDEPENDENT

RNAi innate immunity genome defense

SEQUENCE-SPECIFIC

dsRNA

dsRBD

dsRNA binding domain

other e.g. Staufen-mediated decay

RNA silencing

RNAi & related pathways

The principle of RNA silencing

substrate

AGO

Dicer small RNA production

targeting

silencing (function)

RdRP

AGO

RdRP

amplificiation

uninjected parent not stained

antisense injected m

ex-3

in

situ

hyb

rid

iza

tio

n

dsRNA injected

Canonical animal RNAi pathway

RdRP

Dicer

AGO

AGO

AAAAA

mRNA cleavage

siRNA

dsRNA

siRNA

dsRBP

aberrant RNA

AGO

dicing

slicing

RISC

loading

substrate synthesis

viruses, convergent transcription, inverted repeat transcription, RdRp activity, artificial

Canonical animal RNAi pathway

• defense against elements (viruses, TEs) producing dsRNA • with or without RdRP amplification loop

RNAi: long dsRNA-induced sequence-specific mRNA degradation

dicing

repression

RISC (miRISC)

loading miRNA

Dicer

AGO

AAAAA

inhibition of translation mRNA degradation

GW182

pre-miRNA

dsRBP

AGO

Drosha

pri-miRNA pre-miRNA

DGCR8

Microprocessor complex

EXP5

mRNA degradation

RELOCATION TO P-BODIES

nucleus

cytoplasm

Canonical animal miRNA pathway

DGCR8

• post-transcriptional regulation of gene expression • stoichiometry (miRNA abundance/cell) matters

miRNA: regulatory RNAs produced by Dicer from genome-encoded small hairpin precursors

DICER

small RNA biogenesis

Dicer

RNaseIIIa “platform”

(DUF283)

RNaseIIIb

PAZ

DEXD

HELICc

NSMB 2012 19(4):436-40.

Dicer

• Dicer makes small RNAs

Three distinct small RNA populations Dicer

• predictability of the 5’ end

ARGONAUTE

small RNA loading target recognition

silencing

Argonaute-mediated silencing effects AGO

AGO2

AAAAA

mRNA cleavage by AGO2

Eukaryots

“miRNA-like” mRNA degradation

P-BODIES

CCR4-NOT

AGO

AAAAA

Inhibition of translation/deadenylation

GW182

Eukaryots

“RNAi-like”

RdDM

DNA methylation

Plants

other transcriptional silencing

heterochromatin

e.g. S. pombe … poorly understood in animals

POST-TRANSCRIPTIONAL

TRANSCRIPTIONAL

Argonaute structure and function AGO

PIWI

PAZ

N MID

mRNA

siRNA 3’

5’ 5’ 3’

• Argonaute structure explains principles of target recognition and repression

Argonaute structure and function AGO

PIWI

PAZ

N MID

mRNA

siRNA 3’

5’ 5’ 3’

• Dissociation constants for seed matching targets are in a pM range • low abundant miRNAs unlikely to have significant regulatory effects • seed match + abundance = siRNA off-targeting

Argonaute-mediated silencing effects AGO

Parameters influencing silencing by small RNAs

• small RNA abundance (stoichiometry)

• target site accessibility

• complementarity with the target

• type of silencing (transcriptional/post-transcriptional)

A small RNA seed sequence defines the minimal sequence complementarity required for silencing

OFF-TARGETING

Perfect vs. imperfect basepairing

nucleus cytoplasm

Ago

Class 2 hairpin

(miRNA-like) Class1 hairpin

(shRNA)

siRNA

EXPRESSION VECTOR

.

TRANSFECTION

Ago2

AAAAA Cleavage of

mRNA by Ago2

Ago

AAAAA

mRNA degradation Inhibition of translation

RELOCATION TO

P-BODIES

RISC

loading

short RNAs

(miRNAs and siRNAs)

Argonaute – targeting & off-targeting AGO

• off-targeting is siRNA-specific • any siRNA has off-targeting potential

Jackson et al. (2003) Nature Biotech



Jackson et al. (2003) Nature Biotech

• off-targeting is largely concentration-dependent • it is strongly reduced in sub-nanomolar range

transfected at 100nM

Pooling reduces off-target effects without affecting effciency

siRNA2 siRNA4 siRNA3 siRNA1 pool

Thermofisher/Dharmacom website


• siRNA pooling is a way to reduce concentrations of individual siRNAs while keeping the constant siRNA amount in a transfection

• natural siRNA pools produced from siRNAs are highly specific because of a highly diluted off-targeting effect


• off-targeting potential stems from seed sequence frequency • siRNA knock-downs - usually employ nM concentrations • hydrodynamic transfection (40 mg/mouse – Nature, 418, 38-39)

Ago

AAAAA

miRNA-like Inhibition

of translation

seed = nucleotides 2-7

Ago2

AAAAA

RNAi-like Cleavage of

mRNA by Ago2

seed = nucleotides 2-7

RNA silencing in different organisms

mRNA degradation

RNAi

Dicer

Co-existence of miRNA & RNAi pathways

defense gene control

inhibition of translation

miRNA

Dicer

AGO AGO

Arthropod set up

mRNA degradation

RNAi




miRNA

AGO

Dicer

Annelida set up (some Molluscs?)

mRNA degradation

RNAi



miRNA

AGO

Dicer

interferon response

PKR


defense

Vertebrate set up

mRNA degradation

RNAi




miRNA

AGO

Dicer

RdRP Nematode set up (some Molluscs?)

Nematodes

endoRNAi antiviral defense replication

dsRNA dsRNA

DRH-1

Dicer

gene control

ERGO-1

AAAAA

mRNA cleavage

RdRP

WAGOs

RDE-4

1o siRNA

1o siRNA 26G RNA

22G RNA

DRH-3

2o siRNA

Dicer

immunity

RDE-1

AAAAA

mRNA cleavage

RdRP

SAGO-2

RDE-4

1o siRNA

22G RNA

DRH-3

2o siRNA

ERI

ERI

RDE-8

1o siRNA 22-23 nt

RNA clearance

exoRNAi

RDE-1

AAAAA

mRNA cleavage

RDE-8

RdRP

WAGOs

dsRNA

injection feeding soaking

1o siRNA

1o siRNA 22-23 nt

22G RNA

DRH-3

2o siRNA

DRH-1

Dicer RDE-4

nucleus

cytoplasm

Dicer

ALG-1/2

gene control

AGO

AAAAA


AIN-1

pre-miRNA

miRNA

miRNA 22-23 nt

pri-miRNA


Nematodes

RNA clearance

exoRNAi

RDE-1

AAAAA

mRNA cleavage

RDE-8

RdRP

WAGOs

dsRNA


1o siRNA

1o siRNA 22-23 nt

22G RNA

DRH-3

2o siRNA

DRH-1

Dicer RDE-4

0.5 - 1.0x106 dsRNA molecules per each gonad arm

Tabara et al., 1998

Plants

RDR6

RNA clearance (post-transcriptional)

transgene & viral silencing

AGO

dsRNA

viral long hairpin

21/22 nt siRNA

SDE3

sense RNA

RDR6 SGS3

DCL4/2 DCL3

AGO4/6

24 nt siRNA

RdDM AGO

SDE3

DNA methylation (transcriptional)

HEN1 HEN1 DRB3 DRB4

sense RNA

TAS loci

AGO miRNA

RDR6

DCL4

AGO1/7

tasiRNA

21nt tasiRNA

Gene regulation during development

HEN1 DRB

miRNA pathway in plants & animals

gene control

Plants

mRNA cleavage inhibition of translation

pre-miRNA

HYL1

AGO1 miRNA 21 nt

pri-miRNA

SE

HYL1 SE DC

L1

DC

L1

AGO1

AAAAA

SUO

HEN1 HEN1

nucleus cytoplasm

nucleus cytoplasm

Arthropods

AGO1

gene control

AGO1

AAAAA


GW182

LOQS

miRNA 21-23 nt

Dicer-1

nucleus cytoplasm

Mammals

AGO1-4

gene control

AGO1-4

AAAAA


GW182

TARBP2

miRNA 21-23 nt

Dicer-1

pre-miRNA

pri-miRNA

pre-miRNA

pri-miRNA

DGCR8

DGCR8

Drosha DGCR8

DGCR8

Drosha

Plants

AGO1

DCL2 DCL3

AGO10 AGO7

DCL1

miR-390 miR-156/166 U U A A

AGO2

MAIN miRNA PATHWAY

AGO4/6/9

21 nt 24 nt

long inverted repeats (evolving miRNAs)

ALTERNATIVE miRNA PATHWAY

DCL4

• highly complex RNA silencing system 4x Dicer, 10-20 Argonautes • a number of small RNAs, TGS & PTGS effects

Plants – transcriptional silencing

Canonical RdDM Non-canonical RdDM

• highly complex RNA silencing – crosstalks & redundancy

small RNA mobility

RNAi mobility - systemic RNAi

dsRNA

dsRNA

dsRNA

dsRNA delivery RNAi effect

Cell autonomous RNAi

Systemic RNAi

Environmental RNAi

dsRNA

Example

0.5 - 1.0x106 dsRNA molecules per each gonad arm

mammals

C. elegans some Arthropods (Tribolium) plants

C. elegans insects

Plants – spreading of RNA silencing

short distance

long distance

Plants –> Animals

? ?

?

environmental & systemic RNAi

circulating miRNAs ?

?

Huang et al., 2006

Baum et al., 2007 Mao et al. 2007

environmental & systemic RNAi

Plants –> Animals

Unclear/controversial issues: Mechanism of transport • Mechanism of transport across membranes not explained • Unclear if free or bound to a protein • Survival in digestive tract? Effector complex structure • Would require binding of methylated single stranded RNAs by AGO Targeting stoichiometry • Concentrations estimated 68-250 fM – too low • Authors calculate ~850 molecules per cell, cannot be verified – data not released

Plants –> Animals

• meta-study of xenomiRs of 824 datasets from human tissues and body fluids • xenomiRs commonly present in tissues (17%) and body fluids (69%), • low abundance, 0.001% of host human miRNA counts • no significant enrichment in sequencing data from tissues and body fluids exposed

to dietary intake (e.g. liver). • no significant depletion in tissues and body fluids that are relatively separated

from the main bloodstream (e.g, brain and cerebro-spinal fluids) • the majority (81%) of body fluid xenomiRs stem from rodents, which are rare

human dietary contributions, but common laboratory animals. • body fluid samples from the same studies are clustered by xenomiR compositions

- suggesting technical batch effects. • feeding studies - no transfer of plant miRNAs into rat blood, or bovine milk

sequences into piglet blood.

doi: 10.1261/rna.059725.116

RNA, advanced online, Jan 6., 2017

Key points

• a targeting repertoire of a small RNA is largely determined by its seed – nucleotides 2-8. • not absolute rule (non-canonical binding) • allows some predictability, especially for conserved targets

• RNAi-like cleavage or miRNA-like target repression silencing effects are primarily defined by AGO isoform and basepairing

• targeting efficiency is determined by: • small RNA abundance (stoichiometry) • target site accessibility • complementarity with the target

• vertebrates have lack systemic RNAi, an RdRP amplification system, and highly processive Dicer -> inefficient RNAi

• plant small RNA pathways use 3’ end 2-O-methyl modification of all small RNAs. In mammals, such modification is found only in piRNAs bound to PIWI AGO cladein the germline

CREDITS: Miloslav Nic Tomas Novotny Jan Paces

END

Rana 2007

Sequence-specific RNA silencing

nucleus cytoplasm

GW182 AGO2

AAAAA

Cleavage of mRNA by Ago2

Exportin 5-mediated transport

AGO

AAAAA

mRNA degradation Inhibition of translation

relocation to P-bodies

GW182

miRNA duplex

Dicer

GW182 AGO

RISC loading

Mammalian microRNA pathway

pri-miRNA pre-miRNA

DGCR8


DGCR8

Dicer cleavage

targeting

pre-miRNA

Drosha

miRISC

RNA silencing in selected

taxonomic groups

Task I Mode-of-action of dsRNA and miRNA pathways

Animal Dicer evolution

• RNAi-dedicated Dicer-2 in Arthropods is a derived character

• the mammalian “miRNA” Dicer is related to miRNA-producing Dicer-1 in Arthropods

• Dicer in C. elegans produces efficiently miRNAs and siRNAs

Dicer

“miRNA” Dicer

mRNA degradation

RNAi

Dicer




miRNA

Dicer

AGO AGO

mRNA degradation

RNAi




miRNA

AGO

Dicer

mRNA degradation

RNAi



miRNA

AGO

Dicer

interferon response

PKR


defense

Interferon response induced by long dsRNA (>30bp)

sensing

specific responses

MDA5 TLR3

PKR OAS

RIG-I

common response

INTERFERON RESPONSE

ISG interferon-stimulated genes

eIF2a P

RNaseL

global inhibition of translation

global mRNA degradation

2’,5’-OA

• The interferon response can be detected/monitored

Small RNA pathways in animals

mammals birds fish

Arthropoda

Nematoda

Annelida

Mollusca

Cnidaria

Porifera

Chordata

ECDYSOZOA

LOPHOTROCHOZOA

Chelicerata Myriapoda Crustacea Hexapoda

Trilobita †

Mammals (and vertebrates in general)

OAS

MDA5

TLR3

PACT

Dicer

miRNA RNAi

AGO1-4

gene control

AGO1-4

AAAAA


GW182

AGO2

AAAAA

mRNA cleavage

pre-miRNA

miRNA

dsRNA

siRNA

TARBP2 PKR

OAS

RIG-I

translational repression

RNAse L

IFN signaling

interferons &

interferon stimulated genes

Interferon response

common sensors

RNA silencing

antiviral defense

dsRNA

• miRNA pathway is the main RNA silencing pathway • main dsRNA response = sequence-independent interferon response

Annelids

Dicer

miRNA RNAi

AGO

AGO

AAAAA


AGO

AAAAA

mRNA cleavage

pre-miRNA

miRNA

dsRNA

siRNA

TARBP2 ?

OAS

RIG-I ?

MDA5 ?

RNAse L

signaling

innate immunity?

dsRNA response

common sensors

RNA silencing

dsRNA

?

?

• almost no functional data, set-up seems similar to mammals

Molluscs

RdRP

Dicer

miRNA RNAi

AGO

gene control & antiviral defense?

AGO

AAAAA


GW182

AGO

AAAAA

mRNA cleavage

pre-miRNA

miRNA

dsRNA

siRNA

TARBP2 PKR

OAS

RIG-I

MDA5

translational repression

RNAse L

IFN signaling

interferons &

interferon stimulated genes

Interferon response

common sensors

RNA silencing

antiviral defense

dsRNA

? MX

• almost no functional data, set-up seems similar to mammals • possible RdRP loop – would make it similar to nematodes

Arthropods

nucleus

cytoplasm

AGO2


AGO2

AAAAA

mRNA cleavage

siRNA

dsRNA

PKR

RIG-I

MDA5

Interferon response

common sensors

RNAi

dsRNA

AGO1

gene control

AGO1

AAAAA


GW182

pre-miRNA

LOQS

miRNA

miRNA 21-23 nt

pri-miRNA

Microprocessor

R2D2

Dicer-1

Dicer-2

TLR3?

signaling

innate immunity?

• separated miRNA & RNAi • sensors of the interferon response present

Nematodes

C. elegans is an outstanding model for analyzing RNA silencing • highly complex RNA silencing system • one Dicer but 26 Argonautes and 3 RdRPs • four pathways can be recognized

• miRNA • exoRNAi • endoRNAi • antiviral defense

• primary and secondary RNAs (amplification of the response) • cytoplasmic and nuclear Argonautes • systemic RNAi, sensitive, cheap, temperate areas worldwide

0.5 - 1.0x106 dsRNA molecules per each gonad arm Tabara et al., 1998

Nematodes

nucleus

cytoplasm

Dicer

ALG-1/2

gene control

AGO

AAAAA


AIN-1

pre-miRNA

miRNA

RNA clearance

exoRNAi

miRNA 22-23 nt

RDE-1

AAAAA

mRNA cleavage

RDE-8

RdRP

WAGOs

dsRNA

endoRNAi antiviral defense replication


dsRNA dsRNA

DRH-1

1o siRNA

1o siRNA 22-23 nt

22G RNA

DRH-3

2o siRNA

Dicer

gene control

ERGO-1

AAAAA

mRNA cleavage

RdRP

WAGOs

RDE-4

1o siRNA

1o siRNA 26G RNA

22G RNA

DRH-3

2o siRNA

Dicer

immunity

RDE-1

AAAAA

mRNA cleavage

RdRP

SAGO-2

RDE-4

1o siRNA

22G RNA

DRH-3

2o siRNA

ERI

ERI

pri-miRNA


RDE-8

1o siRNA 22-23 nt

DRH-1

Dicer RDE-4

Plants

AGO1

DCL2 DCL3

AGO10 AGO7

DCL1

miR-390 miR-156/166 U U A A

AGO2

MAIN miRNA PATHWAY

AGO4/6/9

21 nt 24 nt

long inverted repeats (evolving miRNAs)

ALTERNATIVE miRNA PATHWAY

DCL4

• highly complex RNA silencing system 4x Dicer, 10-20 Argonautes • a number of small RNAs, TGS & PTGS effects

miRNA pathway in plants & animals

gene control

Plants

mRNA cleavage inhibition of translation

pre-miRNA

HYL1

AGO1 miRNA 21 nt

pri-miRNA

SE

HYL1 SE DC

L1

DC

L1

AGO1

AAAAA

SUO

HEN1 HEN1

nucleus cytoplasm

nucleus cytoplasm

Arthropods

AGO1

gene control

AGO1

AAAAA


GW182

LOQS

miRNA 21-23 nt

Dicer-1

nucleus cytoplasm

Mammals

AGO1-4

gene control

AGO1-4

AAAAA


GW182

TARBP2

miRNA 21-23 nt

Dicer-1

pre-miRNA

pri-miRNA

pre-miRNA

pri-miRNA

DGCR8

DGCR8

Drosha DGCR8

DGCR8

Drosha

Plants

RDR6

RNA clearance (post-transcriptional)

transgene & viral silencing

AGO

dsRNA

viral long hairpin

21/22 nt siRNA

SDE3

sense RNA

RDR6 SGS3

DCL4/2 DCL3

AGO4/6

24 nt siRNA

RdDM AGO

SDE3

DNA methylation (transcriptional)

HEN1 HEN1 DRB3 DRB4

sense RNA

TAS loci

AGO miRNA

RDR6

DCL4

AGO1/7

tasiRNA

21nt tasiRNA

Gene regulation during development

HEN1 DRB

Plants – transcriptional silencing

Canonical RdDM Non-canonical RdDM

• highly complex RNA silencing – crosstalks & redundancy

Plants – spreading of RNA silencing

Timeline

• extremely large volume of literature • majority not related to the review purpose (RNAi technology,

miRNA biology, innate immunity …)

Pubmed: RNAi OR RNA interference OR miRNA OR microRNA OR dsRNA

Timeline

1990 2000 2010 2015

discovery of RNAi

RNAi mechanism solved (AGO2 crystalized) co-suppression

1st miRNA Let-7

& siRNA

Dicer discovered

Dicer crystalized GW182:AGO2

Single-molecule analysis of AGO binding

pre-RNAi era - mainly plant PTGS research - initial miRNA research

RNA silencing core molecular mechanism

deciphering - mutation screens - biochemical approach

miRNA research

Plant co-suppression, PTGS, VIGS, TIGS etc. mechanisms

RNAi research

Literature review process

1. Searches in bibliographic databases n = 641 975

2. Citation pearl growing using publications known to be landmark publications in the field. (Annex C)

3. Removal of duplicates (compilation of a comprehensive set of scientific and grey literature).

4. Exclusion of references published since 2000 without DOI

5. Filtering for relevance to individual ELS questions

6. Screening of titles and abstracts

7. Study selection based on full-text reports

n = 682 911

n = 239 987

n = 190 734








n = 682 911

n = 239 987

n = 190 734


reference database

Scopus

keyword search

Pubmed WoS

citations of 47 landmark papers covers highly-cited pioneering papers from the pioneering times when

nomenclature was not established and uniformly adopted across the field

ProQuest


double strand* rna, dsrna rna interference, rnai, gene silenc*, ptgs Dicer, rnase III, argonau*, ago1, ago2, Piwi, wago, rde1 or rde-1, r2d2 tarbp2 or trbp2 mirna or microrna, sirna, 21u rna oligoadenylate, Pkr









n = 682 911

n = 239 987

n = 190 734

- described in detail in 2. Data & Methodologies

SPECIFIC SET-UP FOR EACH TASK/ELS QUESTION OR TAXONOMIC GROUP:


MAMMALS BIRDS FISH MOLLUSCS ANNELIDS ARTHROPODS NEMATODES PLANTS

choice of keywords for

reference inspection


references with abstracts with

highlighted relevant

keywords

relevant/irrelevant choice

chosen filtering keywords

exclude

include

Reis et. al (2015)

filtering keywords


publication type annotation

optional annotation buttons


relevant

include

export to Endnote









n = 682 911

n = 239 987

n = 190 734

- described in detail in 2. Data & Methodologies

lack of 3’ overhangs

induces IFN via Rig-I

dsRNA > 30 bp activates PKR and 2’,5’-OAS

some sequence

motifs within ssRNA

can activate IFN cationic lipid-RNA complexes

activate IFN via TLR3 and TLR7

5’ triphosphate introduced by

phage RNA polymerases

activates IFN

siRNA < 30 bp can activate PKR

lack of 3’ overhangs

induces IFN via Rig-I

dsRNA > 30 bp activates PKR and 2’,5’-OAS

some sequence

motifs within ssRNA

can activate IFN cationic lipid-RNA complexes

activate IFN via TLR3 and TLR7

5’ triphosphate introduced by

phage RNA polymerases

activates IFN

siRNA < 30 bp can activate PKR

Interferon response induced by siRNAs

24 h

ou

rs

72 h

ou

rs

mock

siRNA A

siRNA B1

siRNA B2

siRNA C

mock

siRNA A

siRNA B1

siRNA B2

siRNA C

dsRBD Zβ deaminase Zα

NLS NES ADAR1p150

ADAR1p110

ADAR2

ADAR3

Adenosine deamination

Kono & Akiyama, 2013

DOI: 10.5772/55203

nucleus

cytoplasm

ADAR1

degradation

?

AGO

Dicer Dicer AGO

dicing

RISC-loading complex

asymmetry sensing

HSP90

AGO

Dicer

HSP90

Argonaute loading passenger strand removal

Argonaute loading AGO

sense siRNA strand (passenger)

antisense siRNA = targeting (active) strand!

5’-CGUACGCGGAAUACUUCGAdTdT-3’

|||||||||||||||||||

3’-dTdTGCAUGCGCCUUAUGAAGCU-5’

• siRNA duplex undergoes loading of one of the strands on RISC • 5' portion of the selected strand is paired less stably than its 3' portion • ssRNA could reconstitute RISC; 10- to 100-fold higher concentrations required

relative to siRNA duplexes (Martinez et al., 2002, Cell. 110(5):563-74)

Omics and bioinformatics applied to the

characterization of plant materials

24 May 2017 Esther Kok

Acknowledgements

RIKILT Wageningen UR

Jeroen van Dijk

Martijn Staats

Marleen Voorhuijzen

Martijn Slot

Roberta Mariot

Joseph Evaristo

Rico Hagelaar

2

WUR - Biometris

Hilko van der Voet

WUR – Plant breeding

Ronald Hutten

Richard Visser

University of Nijmegen –

Chemometrics

Jeroen Jansen

SAFETY ASSESSMENT OF A NEW GM VARIETY

3

Parent crop

Identity, phenotypic &

agronomic performance

History of safe use

Compositional analysis

Donor, transgene(s) and delivery process

Description of donor

Description of vector DNA

Transgene delivery process

Characterisation of introduced

DNA

Characterisation of insertion site

Characterisation of gene

product(s)

Structure, identity and

characterisation

Mode of action/Specificity

Toxicity

Allergenicity

New GM crop

Identity, phenotypic &

agronomic performance

Nutritional analysis

Compositional analysis

Safety analysis

(animal studies)

Focus: potential presence of unintended effects of the genetic modification

Unintended effects

4

If the DNA code is not clear

If we can not interpret observed changes in the DNA

We use the compositional data

(targeted analyses)

We use the compositional data (targeted analyses

Why may potential unintended effects not be relevant?

• We have a long history of innovative plant breeding with

very few examples of adverse effects

• Plant breeders take their responsibility to develop new crop

varieties that are safe and nutritious

• It is unlikely that a safe variety is transformed into an unsafe

variety as the result of unintended effects

Unintended effects

5




(targeted analyses)


Why may potential unintended effects be relevant?

• A range of new and powerful techniques (Crispr-Cas,

synthetic biology) allow the rapid introduction of new RNAs,

proteins and secondary metabolites, unknown to our food

supply chain, possibly even unknown to nature.

• Because of the targeted and precise techniques plant breeding

programmes are becoming shorter with less time

(years/harvests) to assess new varieties for altered

characteristics

Unintended effects

6




(targeted analyses)


• Hazard identification on the basis of:

o Molecular characterisation

o Phenotypic analysis

o Agronomic performance

o Compositional analysis (targeted analyses)

Unintended effects

7




(targeted analyses)







o Animal feeding trials with whole foods

Unintended effects

8




(targeted analyses)







o Animal feeding trials with whole foods

In the GRACE project:

- animal feeding trials with whole foods

- detailed compositional analyses - same maize materials

Compositional analysis (targeted)

9

Non-GM counterpart

GM variety

Compositional analysis (targeted)

10

Non-GM counterpart

GM variety

Conventional variety 1






Compositional analysis,

targeted vs omics analysis

11

Targeted analyses:

• key nutrients (macronutrients/micronutrients),

• key anti-nutrients, including natural toxins

Omics analyses:

• Transcriptome: all transcribed DNA products (RNA)

• Proteome: all proteins

• Metabolome: all secondary metabolites

Unintended effects

12




(targeted analyses)


Targeted analyses • Few hundreds of end-

points

• Limited coverage of individual metabolic routes

• Advanced data analysis is required (comparison with conventional varieties)

• Natural variation needs to be included!

Omics analyses • Many thousands of end-

points

• Broad coverage of individual metabolic routes

• Advanced data analysis is required (comparison with conventional varieties)

• Natural variation needs to be included!

Omics analyses

13




(targeted analyses)


Omics analyses lead to very large datasets. The question is: how to analyse for meaningful differences in the omics profiles, given the fact that there is much natural variation between plants due to e.g. - Genotype - Environmental conditions of growth (soil and climatological conditions) Model developed with Wageningen UR Biometris (statisticians) and University of Nijmegen, dept of Chemometrics Basic criterium: profiles of varieties that can not be considered as safe should fall outside of the one class

14

Compare transcriptomics profiles

Omics profiles of

commercial crop plants

Build a one-class

classification tool

Classify the new profile

Omics profiles of

commercial crop plants

Build a one-class

classification tool

Classify the new profile

Within the safe one-

class?

Further analysis

No further analysis

Yes No

Omics analysis: one class model (SIMCA)

1 6

Safe


1 7

IN or OUT? Safe Parent GM


GRACE conclusions Unintended effects can likely be more effectively traced by informative

omics analyses compared to animal feeding studies with whole foods:

GRACE data have shown that the comparative safety assessment

(Implementation Regulation 503/2013) can also be adopted for omics

data, e.g. using the one-class model approach: the GM variety can be

compared to its closest conventional comparator, as well as to a range of

conventional varieties.

GRACE data have shown that the one-class model classifies mycotoxin-

contaminated maize samples as outside of the one ‘safe’ class, the

results would provide a scientific basis for further analysis.

GRACE conclusions

• All maize varieties fed to the test animals (90-d) in the course of

GRACE were classified by the one-class model as inside of the one

‘safe’ class

• The one-class model classifies experimental potato varieties that are fit

for human consumption but genetically more distant from the lines that

are currently consumed, in almost all cases as outside of the one ‘safe’

class (indicating that the one-class model represents a conservative

approach)

GRACE conclusions

• Based on these observations: omics data provide qualitatively

structured details of the plant material which facilitates a non-targeted

“safety“ evaluation.

• Thereby it provides a better basis for the decision on the scientific

rationale to frame the subsequent risk assessment steps, which may

include the performance of an animal feeding trial with the plant-

derived whole food/feed

Thank you very

much for your

attention!

[email protected]

23

1

Cartagena Protocol on Biosafety and Synthetic Biology

Boet Glandorf

GMO Office, RIVM

The Netherlands

2

http://www.sbb.uk.gov.in/

3

https://www.google.nl/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjS9dORgPzTAhXHJlAKHUmTD3sQjRwIBw&url=https%3A%2F%2Fwww.slideshare.net%2Fexpattam%2Fwhat-is-the-value-of-biodiversity-16127131&psig=AFQjCNECMLQ6aIxUNmKlhAChOr57PJ5vaQ&ust=1495284502621008

4

Objective Cartagena protocol

5

To contribute to ensuring the safe transfer, handling and use of LMOs resulting

from modern biotechnology that may have adverse effects on the biological

diversity, taking also into account risks to human

health

http://images.google.ca/imgres?imgurl=http://www.bigfoto.com/sites/galery/flowers1/flower-pic_005.jpg&imgrefurl=http://www.bigfoto.com/themes/nature/flowers/&h=788&w=1224&sz=89&tbnid=yYB72nPazzsJ:&tbnh=96&tbnw=149&start=37&prev=/images%3Fq%3Dflower%26start%3D20%26hl%3Den%26lr%3D%26sa%3DN

Cartagena Protocol

6

•Negotiated under the Convention on Biological Diversity (CBD)

• Adopted 29 January 2000 after 4 years of intense negotiations • Entry into force: 9 September 2003 • 170 ratifications/ accessions • 8 meetings of the governing body (COP-MOP)

Scope

7

Applies to: Transboundary movement, transit, handling and use of all LMOs that may have adverse effects on biodiversity, taking also into account risks to human health

http://images.google.ca/imgres?imgurl=http://www.bigfoto.com/sites/galery/flowers1/flower-pic_005.jpg&imgrefurl=http://www.bigfoto.com/themes/nature/flowers/&h=788&w=1224&sz=89&tbnid=yYB72nPazzsJ:&tbnh=96&tbnw=149&start=37&prev=/images%3Fq%3Dflower%26start%3D20%26hl%3Den%26lr%3D%26sa%3DN

How does the Protocol work?

8

The Protocol establishes rules and procedures to

regulate the movements of LMOs from one country to

another

Categories of LMOs

9

•LMOs for intentional introduction into the environment (such as seeds and live fish)

•LMOs intended for direct use as food, feed or processing, LMOs-FFP (such as agricultural commodities – corn, canola and cotton)

•LMOs for contained use (such as bacteria for laboratory scientific experiment)

Procedures for Transboundary Movements of LMOs

Two key procedures:

– The Advance Informed Agreement (AIA) procedure

– Procedures for LMOs intended for direct use as food, feed or for processing (LMOs-FFP)

Precautionary Approach

Objective: Safe Transfer, Handling and Use of

LMOs

Biosafety Clearing-House (BCH) , Capacity-Building,

Compliance and COP-MOP

Supporting Mechanisms:

• Risk

Assessment

• Risk

Management

•Information

Sharing

•Public

Awareness &

Public

Participation

• Rules/

Procedures:

- AIA Procedure

- Procedure for

FFP

• Decision -

making

•Handling,

Transport,

Packaging and

Identification:

- Documentation

for Shipment

- Standards

Key Provisions of the Protocol

12

Regulation (EC) 1946/2003 regulates transboundary movements of GMOs and transposes the Cartagena Protocol on Biosafety into EU law The Protocol sets common rules for the trans-boundary movement of Living Modified Organisms to ensure the protection of biodiversity and human health globally. The Regulation, which addresses in particular exports of GMOs, obliges EU countries to take legal, administrative and other measures to implement their commitments under the Protocol. It establishes the procedures for the trans-boundary movement of GMOs including: - notification to importing parties - information to the Biosafety Clearing House - requirements on identification and accompanying documentation

http://eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX:32003R1946

https://ec.europa.eu/food/plant/gmo/international_affairs_en

Cartagena protocol Main discussion items at last COP MOP meetings were:

• Adoption/endorsement of Guidance on Risk Assessment

of LMOs (Road map)

• Development of further RA guidance for specific groups

of LMOs, such as LM fish and organisms obtained by synbio

13

Synthetic biology: new and emerging issue under the CBD?

2012 Decision XI/II New and Emerging issues

Noting, based on the precautionary approach, the need to consider the potential positive and negative impacts of components, organisms and products resulting from synthetic biology techniques on the conservation and sustainable use of biodiversity, requests the Executive Secretary, subject to availability of resources…

● Compilation of developments in synbio

● Synthesis of information on synbio

● Review by technical body (SBSTTA)

● Is synbio a new and emerging issue?

14

Synthetic biology: new and emerging issue under the CBD

2014 Decision XII/24. New and emerging issues: synthetic biology?

● Coordinated approach synthetic biology and Cartagena Protocol

● SBSTTA not clear if synbio is new and emerging issue

● Precautionary approach: only introduction after risk assessment, risk management is in place

● Establishment of Ad Hoc Technical Working Group (AHTEG) and online forum

● Mandate: operational definition synbio, difference synbio and LMO, benefits and risks synbio, best practices for risk assessment and monitoring, framework to address impacts

After review by SBSTTA, draft decision to be discussed in COP 2016

15

General Surveillance van GGO's in Nederland | 27 september 2010

16

3 opinions in total

17

https://www.cbd.int/cop2016/

Synthetic biology: new and emerging issue under the CBD

2016 Decision XIII/17 New and emerging issues: synthetic biology

• Precautionary approach: only introduction after risk assessment, risk management is in place, also applies to organisms with a gene drive

• Current applications synbio are LMO

• Risk assessment methodology LMOs is applicable to synbio

• Unclear of some organisms obtained by synbio in the future will fall under definition of LMO

• Collection of further info on experience with synbio, such as risk assessments, effects (positive, negative)

• Extension AHTEG, online forum

• Mandate: to review recent technological developments, synbio organisms that are no LMO, best practices, detection and monitoring

18

Cartagena protocol

Cancun 2016 Draft decision

• Adoption of Guidance on Risk Assessment of LMOs

• Development of further RA guidance for specific groups

of LMOs, such as for organisms obtained by synbio

2016 Decision VIII/12

• Take note of Guidance as one of the Guidances

• No extension of the AHTEG on Risk Assessment

• No development of further guidance on LM fish and synbio

• Compilation of views on:

- topics for further guidance and

- - criteria to decide when such guidance is considered necessary

19

http://www.cbd.int/doc/meetings/bs/mop-08/official/bs-mop-08-08-add1-en.pdf

https://www.cbd.int/cop2016/

Next step

Outcome of:

● Online forum Cartagena Protocol

● Online forum synbio

● AHTEG synbio

will be discussed in technical body (SBSTTA) in 2018

Decisions will be taken in COP MOP (Cartagena Protocol) en COP (synbio) at the end of 2018 in Egypt, based on report SBSTTA

20

21

Thanks!!

Documents

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