Centre for Integrated Research in Biosafety

Tel:+6433642500,Fax:+643642590 Email:jack.heinemann@canterbury.ac.nz

EvaluationofrisksfromcreationofnovelRNA moleculesingeneticallyengineeredwheatplants andrecommendationsforriskassessment

AnexpertopinionofProfessorJackA.Heinemann,PhD 28August2012

Onthe12thJuly2012IwascontactedbytheSafeFoodInstitutewitharequest toprovideanexpertscientificopiniononGMwheatresearchbeingconducted bytheCSIRO.Includedinthiscorrespondencewastheadditionalrequestthat: Ifyoudobelievethattherearerisks,canyoupleaseproviderecommendations foranystudiesthatwouldprovideinformationtoconfirmorexcludeanyrisksto humanhealth. Thefollowingismyexpertopinionaboutwhetherplantsgeneticallymodifiedto produceanRNAinterference(RNAiorcosuppression)effectmaycreatearisk forhumanhealthortheenvironment. Iamamolecularbiologist.IhavebeenanacademicattheUniversityof Canterburysince1994.Priortothat,IwasemployedbytheUSNational InstitutesofHealth.MydoctoratewasconferredbytheUniversityofOregonat Eugene(1989)andmyBachelorofScience(withhonours)degreefromthe UniversityofWisconsin,Madison(1985).Iaminvolvedinriskassessment researchandparticipateinriskassessmentthroughevaluationofassessments providedtoregulatorybodiesandthroughthedevelopmentofinternational guidancedocumentsforriskassessment.Ihaveover100scholarlyworks publishedonthetopicofmolecularbiology,genetics,riskassessmentandother scientificmatterswithinmyexpertise.Ipublishinleadinginternationaljournals andmyworkhasbeenrecognisedbyprestigiousprofessionalorganisationsfor itsexcellence. IwillprovideashortoverviewofhowdsRNAmediatedsilencing(viaRNAi, cosuppressionandothersimilarpathways)worksandhowitismanipulated throughgeneticengineering.Moreextensivecoveragewithrelevancetorisk assessmentcanbefoundinAppendix1ofHeinemann(2009).Iwillfocusonthe riskpathwaysthatIbelieveareplausibleandrelevant.Potentiallycritical technicaldetailsoftheactualconstructsusedinthewheatthatwerebeing trialledwerenotavailabletome.Iunderstandthatthesedetailsarenotinthe publicdomain1.Thisanalysisthereforeisbasedonthepotentialformodifications thatcouldcauseharmfromtheviewpointofascientistattemptingtominimise typeIIerrors,i.e.,thosethatwouldfalselyfindnoriskwhenoneindeedexists.

1AsearchofCommonwealthScientificandIndustrialResearchOrganization(CSIRO)patentsintheUSand

EuroperevealedtworelatedpatentsontheuseofdsRNAtocontrolgeneexpressioninplants(US 8,183,217B2,EP2333061A1),butneitheroftheselistedspecificDNAsequencesforSEgenes.

Background: Thechangeintendedtobeintroducedintothegeneticallymodified(GM)wheat throughgeneticengineeringwastheproductionofnovelRNAmoleculesthat turnofftheexpressionofgenes;thesearecalledregulatoryRNAs(atypeof noncodingRNA).ThevastmajorityofexistingcommercialGMplants(e.g., herbicidetolerantorinsecticidetraits)arenotintendedtomakeRNAmolecules thatareinvolvedingeneregulation.Thistypeofmodificationisthereforerare andhasnotbenefitedfromextensiveorvalidatedsafetytestingprocedures. ThereforemyfocusandspecialinterestisontheRNAlevelchanges.

Definitions NoncodingRNAisallRNAinacellthatisnotdirectlyusedasacofactortoorderamino acidsduringproteinsynthesis(i.e.,notmRNA). dsRNAisdoublestrandedRNA.Thestrandsareheldtogetherbyhydrogenbondsbetween nucleotidesinawayanalogoustoDNA.

ThereisatemptationtothinkofgeneticengineeringastinkeringwithDNA.All commercialisedGMplantsatthistimearecreatedthroughinvitroDNA modification.However,notallofthemarecreatedwiththeintentiontoproduce anewprotein.AgrowingminorityaredesignedtochangetheirRNAcontent. ThereasonforthisisthefindingthatRNA,specificallydoublestrandedRNA (dsRNA),isanimportantregulatorofgeneexpression(Appendix1of Heinemann,2009). ThosewhohavestudiedmolecularbiologyknowthatRNAisanintermediate moleculeusedinthecellularreactionsoftranslationtosynthesiseproteins.The mostfamiliarformofRNAismRNA,thesinglestrandedmessenger. Nevertheless,typesofdsRNAshavebeenknownforalongtime.Forexample transferRNA(tRNA),whichisalsousuallytaughtinthefirstencounterwith molecularbiology,hasdsRNAregions.However,itisonlyinthelast1015years thatsmalldsRNAmoleculeshavebecomeknownfortheirroleinregulatinggene expression(HutvgnerandSimard,2008). dsRNAsarevariouslycalledsiRNA(shortinhibitoryRNA),miRNA(microRNA) ormicroRNAlikeRNAandsoonandarefoundationsubstratesinbiochemical pathwaysthatcauseRNAi(RNAinterference),PTGS(cosuppression,post transcriptionalgenesilencing)andTGS(transcriptionalgenesilencing).Inshort, RNAi,PTGSandTGSarecausedbygenesilencing:disruptingtheconnection betweengenesandtheproductionoftheproteinsspecifiedbygenes2.
Definitions siRNAisaprocessedlineardsRNAmoleculewherethetwostrandsareheldtogetherthrough intermolecularbasepairs. miRNAisprocessedsinglestrandedRNAwithadsRNAregionheldtogetherthroughintra molecularbasepairs.miRNAlikemoleculesderivefromsiRNA. RNAi,PTGSandTGSrefertogenesilencingbroughtaboutthroughproductionoforexposure todsRNAmolecules.Thegenesilencingiscausedbyinhibitionoftranslation,mRNA degradation(Figure2),or(TGS)methylationofDNA(andmodificationofhistones).

http://www.nature.com/nrg/multimedia/rnai/animation/index.html.
Heinemann:EvaluationofrisksfromcreationofnovelRNAmoleculesingeneticallyengineeredwheat plantsandrecommendationsforriskassessment

2Foranexcellentanimation,see

dsRNAsformwhenbothstrandsofaDNAmoleculearetranscribedtosynthesise complementaryRNAmolecules(whichthenbindtogetherinthesamewayas DNA),orwhenstretchesofintramolecularcomplementaritycreatestemloop structures(Figure1).AlongdsRNAmolecule(e.g.,prematuremiRNA)is processedintoashorterdsRNA(e.g.,miRNA)andthenonestrandisretained theguidestrandtodirectproteincomplexestotargetmRNAmoleculesand preventtheirtranslation(Figure2),ortotargetandchemicallymodifyDNA sequencesbyadditionofmethylgroupsandcausemodificationofDNA associatedhistoneproteins.Thisprocessisknowntoinhibittranscriptionandto seedheterochromatinformation(Ahlenstieletal.,2012,GrewalandElgin,2007, ReyesTurcuandGrewal,2012,ZhangandZhu,2012). Onceasilencingeffectisinitiated,theeffectmaybeinherited.Thebiochemistry ofthisprocessvariesdependingonorganismandremainsanareaofactive researchwithmanyunknownaspects.Nevertheless,itisknownforexamplethat humancellscanmaintainthemodificationsnecessaryforTGS,creatingactualor potentialepigeneticinheritancewithintissuesandorganisms(Hawkinsetal., 2009). Unintendedgenesilencingisacommonoutcomeofthegeneticengineering process.Indeed,mostcellsinitiallyengineeredusinginvitronucleicacid techniquesultimatelysilencethegeneinsertedbecauseoftheengineering associatedproductionofdsRNA(DenliandHannon,2003,Weldetal.,2001).The newRNAsequencemaybecreatedwhentheDNAstrandnotnormallyusedasa cofactorfortranscriptionisusedassuch(perhapsbecausetheinserthada crypticpromoteractivityorinsertednearapromoter).Theresultingsingle strandedRNAmaybindtothetargetmRNAtocreateregionsoflineardsRNA thatcanbeprocessedintosiRNA(Figure1).Anotherpossibilityisthattheinsert contributestotheformationofastemloop(alsocalledhairpin,shorthairpin (sh)andpanhandleRNA)3,fromwhichthestemmaybeprocessedintoan miRNAlikemolecule(Figure1). dsRNAsareremarkablystableintheenvironment.Insectsandwormsthatfeed onplantsthatmakedsRNAcantakeinthedsRNAthroughtheirdigestivesystem, whereitremainsintact(GordonandWaterhouse,2007,Maoetal.,2007). WormscanabsorbdsRNAthroughtheirskinwhendsRNAissuspendedinliquid (CogoniandMacino,2000,Tabaraetal.,1998).Oncetakenup,thedsRNAcan circulatethroughoutthebodyandaltergeneexpressionintheanimal(Melloand ConteJr.,2004).Insomecases,thedsRNAtakenupisfurtheramplifiedorcauses asecondaryreactionthatleadstomoreanddifferentdsRNAs(secondary dsRNAs)withunpredictabletargets(Baumetal.,2007,GordonandWaterhouse, 2007). TheconcernIwasaskedtocommentoniswhethersiRNAsdesignedtosilence theSEIandSEIIgenes(theSBEIandSBEIIproteins)ofwheatandbarleymight haveofftargeteffects,particularlywhenthesenoveldsRNAmoleculesenterthe humanfoodsupply.FromwhatisknownaboutthebiochemistryofdsRNA mediatedsilencing,andthechemistryofRNA,itisclearthatgenetically engineeredchangesattheRNAlevelcanhaveimportantimplicationsonboth
3E.g.,seelanguageinCSIROpatentUS8,183,217B2.

Heinemann:EvaluationofrisksfromcreationofnovelRNAmoleculesingeneticallyengineeredwheat plantsandrecommendationsforriskassessment

theGMwheat(asintended,andotherwise)andotherorganismsexposedtothe wheat.
Definitions Offtargeteffectsarethosethatresultinunintendedsilencingofother(nottargeted)genes.

Figure1:sourceofnewdsRNAmoleculesfromgeneticengineering. (A)RegardlessofthesourceoftheDNAinserted(dashedblueandgreenlinesin theblackdoublestrandedDNAmolecule)intoagenomebygeneticengineering, itcreatesnewsequences.TheDNAusedwillcreatenewsequencesbecauseit willbebordered(boundarybetweendashedandsolidlines)bydifferent sequencesthaninthesourcegenomebytheengineeringprocess,ormaybe sourcedfromagenomethathasnoorfewsequencematches.(B)Transcription willproducenewRNAmolecules(redanddashedblueandgreenlines)that mightbeabletoformdsRNAbecauseofcomplementarityor(C)becauseof internalbasepairingcausingstemloopstructurestoform(basepairing illustratedwiththinblackconnectinglines).(D)Thismayleadtointendedand offtarget(redlinewithpurpletargetsection)genesilencingintheGMOorin organismsthateattheGMO.

Heinemann:EvaluationofrisksfromcreationofnovelRNAmoleculesingeneticallyengineeredwheat plantsandrecommendationsforriskassessment

Figure2:translationandthetwopathwaysofcytoplasmicdsRNAmediated silencing. Translationofproteins(top)istheprocessoflinkingindividualaminoacids(the subunitsofproteins)intoapolymer,calledapeptide(blueribbon).Thecentral enzymaticactivityisprovidedbytheribosome,amultiproteinandRNA complex(brown),whichusesthemRNA(red)asacofactorandaparticular sequence,thestartcodon,astheplacetobeginproteinsynthesis.Specific dsRNAsareprocessedthroughacomplexbiochemicalpathwaytocreateshort singlestandsthatinanassociatenucleoproteincomplexblocktranslationby directocclusionoftheribosome(bottomleft)orbyrecruitingenzymesthat depolymerisethemRNA(bottomright).

Heinemann:EvaluationofrisksfromcreationofnovelRNAmoleculesingeneticallyengineeredwheat plantsandrecommendationsforriskassessment

I. ItisreasonablefortheplantgeneratednovelRNAmoleculestobe consideredrelevanttoahumanhealthriskassessment. (i) PlantderivedmicroRNAprecursorshavebeendetectedinhumanblood. Thisdemonstratesthattheycansurvivedigestionandbetakenupviathe gastrointestinaltract(Zhangetal.,2012b).PlantderivedmicroRNAsare chemicallyandstructurallysimilartosiRNAconstructsintendedtobe producedinthewheatandthustheircharacteristicsarepredictiveofthe characteristicsofthesiRNAconstructs.ThereisstrongevidencethatsiRNAs producedinthewheatwilltransfertohumansthroughfood. (ii) ThosedsRNAsthathavebeenshowntotransmitviafoodarestable throughcookingandatpH2.0foratleast6hours(Zhangetal.,2012b). ThereisstrongevidencethatsiRNAsproducedinthewheatwillremainina formthatcantransmittohumansevenwhenthewheathasbeencookedor processedforuseinfood. (iii) TheseplantderiveddsRNAmoleculessilencedanendogenousgenein humantissueculturecells,andinvivoinmiceliver,smallintestine,and lung(Zhangetal.,2012b).Thereisstrongevidencethatoncetransmitted, siRNAproducedinwheatwouldhavethebiologicalcapacitytocausean effect. Therefore,itispossiblethatnoveldsRNAmoleculescreatedthroughthegenetic engineeringofthewheat,ormadeatconcentrationsuniquetothiswheat,may bestablethroughstorage,cookingandprocessingwhenusedasahumanfood, andthentransmitthroughfoodorinhalationtohumansandhavethepotential tocauseadverseeffects.Theseeffectsmaybethroughacuteexposureleadingto acytoplasmicRNAieffect(Figure2)oranuclearTGSeffectfollowingexposure. Notably,littleisknownaboutthenumberofofftargeteffectsthatarepossible. Forexample,aTGSeffectarisingfromchromatinremodelingeventsmay representanovelmodeofofftargeteffect(p.2994Hawkinsetal.,2009)and considerablyextendtherangeofofftargeteffects. Wheatisoneofthehumanityslargestsourcesofcaloriesandnutrition.It contributes530kcal/capita/dayand16gofprotein/capita/daytotheworld foodsupply(FAOSTAT,2007)4.Thisisupfrom410kcal/capita/dayand13gof protein/capita/dayin1961(firstyearofFAOSTATstatistics),andsteadywith riceasaproteinandcaloriesource(about20%ofdietarycaloriesnotcounting alcohol)5.Australiaisthe9thlargestproducerofwheatandcontributesan estimated3%toworldproduction.Australiaisconsistentlyoneofthetop5 exportingnations,contributingbyweight11%toworldtradeinwheatand wheatflour,and13%inbarley(FAOSTAT,2009).Withoutquestion,changesto wheatandbarleyinAustraliaeithergoodorbadareimportanttoboth Australiansandtotherestofhumanity. II. ItisreasonablefortheplantgeneratednovelRNAmoleculestobe consideredrelevanttoanenvironmentalriskassessment. (i) SpecificsiRNAscanbetoxicandthetoxicitycanbetransmittedthrough foodtoanimalsofenvironmentalrelevance.Thiswasdemonstratedwhen 4FAOSTATisadatabaseoftheUnitedNationsFoodandAgricultureOrganisation. 5Barleycontributedonly7kcal/capita/dayand0.2gprotein/capita/dayin2007.
Heinemann:EvaluationofrisksfromcreationofnovelRNAmoleculesingeneticallyengineeredwheat plantsandrecommendationsforriskassessment

GMmaizeandcottonplantswereengineeredtoexpressnovelsiRNAsthat wereintendedtobetoxictotargetinsects(Baumetal.,2007,Gordonand Waterhouse,2007,Maoetal.,2007).ThetoxicitywasduetothedsRNA beingtransmittedfromplanttissuestotheinsectsbyingestion,andthen beingfurtherprocessedintosiRNAthatsilencedoneormoregenes essentialforlife,oressentialfordetoxifyingnaturalplanttoxins(i.e., gossypolincotton).ThereisstrongevidencethatsiRNAsproducedinthe wheatwilltransfertorecipientorganismsintheenvironment. ThisriskisknowntotheCommonwealthScientificandIndustrialResearch Organization(CSIRO),theGMwheatdeveloper,becauseitholdsapatentto transmitdsRNAthroughfoodtoarthropodsandtocausegenesilencingin theanimals6.Initspatentapplication,theCSIROmakesclaimtoaprocess fordeliveringdsRNAthroughfeedingatransgenicorganismexpressing thedsRNAtothearthropod.Thetransgenicorganismisselectedfrom,but notlimitedto,thegroupconsistingof:plants,yeast,fungi,algae,bacteriaor anotherarthropodexpressingthedsRNA. (ii) UnintendedsecondarydsRNAsthatmightbegeneratedinplantaorin animalsconsumingtheplantcannotbeanticipatedbutmaywellexist. ThesesecondarydsRNAsmayhavegeneregulatoryactivitiesandthusact likesiRNA.ThismeansthatdsRNAscreatedbythegeneticengineeringof wheatmaycausetheproductionofadditionalunintendedorunanticipated dsRNAmoleculesinboththegeneticallyengineeredwheatandinany organismthatconsumesthewheat.Anyoftheseunintendedsecondary dsRNAmoleculescouldbethecauseofanadverseeffect. Importantlyaswell,RNAicanbetransmittedacrossplanttissues regardlessofwheretheinterferenceisinitiallygenerated(Jorgensen,2002, Klahreetal.,2002,Yooetal.,2004).ThismeansthattheintroducedsiRNAs maynotbeconfinedtotheintendedtissue(e.g.,endosperm)andthatthe entireplantneedstobeusedfortestingtoxicitytoindicatorbirds, mammals,insectsandnematodes.Thereisevidencethatunintended secondarysiRNAspotentiallycouldbeproducedeitherinthewheatasan unavoidableoutcomeofthemodification,orinorganismsexposedtowheat tissuesduringcultivationorstorage. (iii) ItisrelevantandnoteworthythatotherregulatoryagenciesinAustralia describethepathwaysthroughwhichRNAiarisesaspoorlyunderstood (FSANZ,2009).Insuchcases,anyriskassessmentwouldhaveahigh uncertaintyastothelevelofriskespeciallyfromunanticipatedeffects.

Wheatcropscover29%ofAustraliasagriculturalarea,whichis53%of Australiassurface.Wheatandbarleycombinedcover37%ofagriculturallandat about17.6millionhectares(FAOSTAT,2009).Achangeinthesecropswith unanticipatedenvironmentaleffectscouldhavelargescaleconsequences. Introduction: TheAustralianCSIROhasconstructedgeneticallymodifiedwheatvarieties intendedtonotexpressSEIonlyintheendospermbecauseofaninducedRNA interference.Barleyvarietieshavebeenproducedthatareintendedtoexpress neitherSEInorSEIIintheendosperm.TheRNAiisintendedtobecreated 6EP2333061A1
Heinemann:EvaluationofrisksfromcreationofnovelRNAmoleculesingeneticallyengineeredwheat plantsandrecommendationsforriskassessment

throughtheintroductionoftransgenesconstructedtoproducesubstratesforthe endogenousdsRNAprocessingpathwaysinplants.Tomyunderstanding,these constructsinvolvetandemrepeatsoftwosequences,withthesecondsequence beingintheoppositeorientation(i.e.,invertedrepeat)tothefirst.Thisallowsfor intramolecularbasepairingandencouragestheformationofshorthairpin dsRNA(Figure3).Therepeatedsequencesarepresumablyexonicsequences fromSEIandSEII,respectively,separatedbyintron3ofSEI.Theactual sequencesusedarenotknowntome(seeabove).Theconstructsareintendedto beprocessedthroughcanonicalsplicingpathwaystoremoveintron3and increasetheefficiencyofprocessingtheresultingdsRNAintosiRNA. SEgenesencodetheSBEstarchbranchingenzymes.Theseenzymescreate16 linkagesthroughcleavageof14linkagesonlinearchainsofglucose.Similar genesarefoundinmanyotherorganisms,includinghumans.Thesimilaractivity toSEIinhumansisencodedbyGBE,theglucan(1,4alpha),branchingenzyme1.
Definitions IntronisaregioninanmRNAmoleculethatisremovedpriortotranslationthroughreactions calledsplicing.

5 3 3 5

intron 3 3 5

3 5

DNA

RNA

Figure3.HypotheticalstructuresofregulatoryRNAmoleculesinGM wheat. Arrowsindicaterepeatedsequencesandtheirorientation.Thenumbers5 and3indicatestrandpolarity.Thegreylinelabeledintron3isthethird intronofSEI. ImakeapreliminaryconsiderationofwhethersiRNAsgeneratedtosilenceSEI mighthavesilencingactivityinhumans.Notethatthisisaconservativeinitial appraisalforseveralreasons. First,theactualsiRNAsequenceswerenotavailable.Thus,Iconsiderwhether anysiRNAsequencescouldhaveaneffect,notwhetherallpossiblesiRNAswould haveaneffect. Second,unintendedsecondarysiRNAsthatmightbegeneratedinplantaorin humanscannotbeanticipatedbutmaywellexist.Forexample,itwassecondary dsRNAsthatweretheeffectiveagentsinestablishingRNAiininsectpestswho
Heinemann:EvaluationofrisksfromcreationofnovelRNAmoleculesingeneticallyengineeredwheat plantsandrecommendationsforriskassessment

ateGMcornplantsdesignedtoproduceprimarydsRNAmolecules(Baumetal., 2007),anditwasprematuremicroRNAofriceplantsmodifiedbyhumancells thatcausedsilencingofgenesinhumantissueculturecells(Zhangetal.,2012b). Moreover,siRNAsdesignedagainstthecodingregionofatargetmayalsofind unintendedbindingtargetsintheintronsofprematureRNAinthenucleus (Seinenetal.,2011).Althoughintronsmaynormallyberemoved,therearemany knownexceptionsandremovalcandifferbetweentissuesandtimeof development,sointronsremainpotentialtargetsofsiRNAs. Third,bioinformatictoolsarenotdefinitiveforpredictingtheeffectivenessof siRNAsatcausinganRNAieffect,particularlyforrulingoutaneffect.Asa biosafetyscientist,IaminterestedinminimisingtypeIIerrors(thosethatresult infalsenegativeidentification)ratherthanminimisingtypeIerrors(thosethat resultinafalsepositiveidentification).AnypossiblesiRNAidentifiedthrough bioinformaticstechniquesisacandidateforfurthertesting,butbioinformaticsis notasubstituteforotherkindsoftesting. Fourth,notallavailablebioinformaticstechniqueswereusedtocreatethisinitial appraisal.Amorerefinedlistofcandidatesmightbefoundusingevenmore sophisticatedandspecialisttechniques(Birminghametal.,2006).However, thesetechniquesweresufficienttoidentifypossibleunintendedtargets. Finally,thebioinformaticsparametersusedbelowhavenotallcomefroma studyofofftargeteffectsinhumans.Humansmaybemoreorlessreactivetothe samesiRNAs.SinceIamabiosafetyscientist,myconcernistominimisetypeII errorsandthusIassumethathumansareatleastasresponsiveasanyresearch organismuntilprovenotherwise.Theliteraturehasabiastowardreporting siRNAswithlargeeffectsratherthansystematicallycataloguingalleffects.This contributestoaperceptionoffewerthanactualunintendedeffects.SinceagainI amminimizingtypeIIerrors,Iassumethateffectsmaybesmallbutbiologically relevantunlessprovenotherwise. IconsiderasrelevantdsRNAmediatedofftargeteffectsthatresultinRNA cleavage,translationalinhibition(cosuppression),ortranscriptionalsilencing (TGS). Methods: GBEandSEIsequenceswereaccessedfromtheNCBIdatabase7.Thesequences werecomparedformatches.Areaswithahighdensityofidentitywerethen evaluatedforpotentialtobetargetedbythesamesiRNA. Criticalassumptions: InformationforthisreportwastakenfromtheOfficeoftheGeneTechnology RegulatorspublicationDIR093Limitedandcontrolledreleaseofwheatand barleygeneticallymodifiedforalteredgrainstarchcomposition.8Thatreport providedthenameofgenestargetedforsilencingandtheoriginingeneralterms ofthesequencesthatwereusedtoconstructtheeventthatisexpectedto expresstheintendedsiRNAs. 7NCBIReferenceSequence:NM_000158.3;GenBank:AF525764.1 8http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/content/dir093
Heinemann:EvaluationofrisksfromcreationofnovelRNAmoleculesingeneticallyengineeredwheat plantsandrecommendationsforriskassessment

Sequenceanalysiswasperformedusingpubliclyavailableaccessionsonthe NCBIdatabase.TheDNAsequencesofthenamedgenesmayormaynotbe exactlythesameasinthewheatvarietiesusedbyCSIRO.However,thisisthe bestpossiblesourceofinformationbarringaccesstotheactualcommercialin confidencesequences.AbsentforthisanalysisweretheDNAsequencesusedby CSIROtoconstructthesiRNAs.Thisproprietaryinformationisnecessaryto conductadefinitivebioinformaticsanalysisforriskassessment.Tocompensate forthis,allpossibleknownsequencesfromthenamedsourcegenesasavailable inNCBIdatabasewereused. Therefore,thisanalysisprovidesanestimationoftheplausibilitythattheGM wheateventcouldcauseanadverseeffectviatheexpressionofthesenovel dsRNAmolecules.Theresultsneitherimplythatallpossibletargetsofinterest havebeenidentified,northatalltargetsidentifiedarecertaintobesilenced.For thesefinalhypothesestobetested,theactualsequenceswouldberequired.It wouldbeincorrecttoassumethatiftargetsdiscussedbelowwerenotaffected, thattheconstructsweresafeforuse.Likewise,itwouldbeincorrecttoconclude thatsequencesimilaritybetweenactualsiRNAsandanytargetsbelowwill necessarilycausethetargetstobesilenced.Moreover,nobioinformaticsbased analysiswillbecapableofmakingadefinitivefindingofsafety.However,itcan provideevidencethatassistsinfocusingexperimentaltestingforpotential adverseeffectsandinthatwaycontributetotheriskassessmentandreduce uncertaintiesbuiltintoregulatorydecisions. Evaluationcriteria: 1. 2. PerfectsequencematchesindicatehighprobabilityofRNAi(thatis~21/21 matches). Shortmatchescancauseofftargeteffects(Linetal.,2005).Inconclusion, 15nucleotides,andperhapsasfewas11contiguousnucleotides,of sequenceidentityaresufficienttodirectsilencingofnontargetedtranscripts andthereforeofftargetgeneregulationcanoccurasaresultofdegradation ofmRNAtranscriptswithpartialidentitytothesiRNAsequence(p.636 Jacksonetal.,2003). Approachingorexceeding95%identityover40nucleotidesispredictedto causeRNAi(Rualetal.,2007). Short(7contiguous9)identicalmatchesinthe3untranslatedregion(UTR) ofmRNAcanbemoredeterminativethannumberofmatchesoverall (Birminghametal.,2006).

3. 4.

Results: TheSEIsequenceusedinthisevaluationwasoriginallysubmittedtoGenBankby theCSIRO.ItincludesthecodingsequenceforSEIandaprecedingpseudogene. SinceIdonotknowwhatsequencewasusedbyCSIRO,theentiresubmitted sequencewasincludedinthisanalysis.

9Theseresearchersfoundthatsurroundingsequences,perhapsthroughtheireffecton2structureofthe

mRNA,accentuatedthestrengthofthesiRNAs.Thecontextdependentsilencingmediatedbypartial complementationbetweenasiRNAanditsunintendedtargetsmakesitmoredifficulttopredicttheoff targeteffectofagivensiRNA(p.4534ofLinetal,2005). Heinemann:EvaluationofrisksfromcreationofnovelRNAmoleculesingeneticallyengineeredwheat plantsandrecommendationsforriskassessment

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ThehumanGBEandplantSEIgenesareverysimilar(Appendix1).Thereis enoughidentitythroughoutthecomparedsequencestopreventrulingout possiblesilencingofthehumanGBEbysiRNAsgeneratedagainstSEI.For example,intheregionofnucleotides9871024oftheGBEopenreadingframe, and2136321400withintheopenreadingframeofSEI,thereare32matches (86%identity),withastretchofonemismatchinarunof21contiguousbases, and16outof16contiguousbasesmakingperfectmatches.Intheregionof nucleotides16941730oftheGBEopenreadingframe,and2207622112within theopenreadingframeofSEI,thereare37matchesoutof40contiguous nucleotides(93%identity),madeupof14matchesin14nucleotides,5matches in7,16matchesin16andafinal2matchesoutof3contiguousnucleotides (Figure4).

Figure4.HighdensityidentityrelationshipbetweenSEIandGBE. Shownarenucleotides16871733ofGBE(query)and2206922115ofSEI (subject).Verticallinesindicateidentity. SEIwasalsocomparedtotheentirehumangenome(Appendix2).Multiple matchesof21contiguousnucleotideswerefound(see,e.g.,horizontalred arrowsindicatingsequencematchesonpages6/770,11/770,1516/770, 31/770,3839/770,43/770and6265/770)10.Thus,theremaypotentiallybe otherunintendedsilencingeffectsdependingonthesiRNAsusedintheGM wheat.

ShortsequencesofperfectidentitybetweenansiRNAandthe3UTRregionof unintendedmRNAsarealsopredictiveforcreatinganRNAieffect.Sequence matchesfromthemRNAofSEIwerethuscomparedtothe3UTRregionofGBE (Table1).ExceptforoneentryinTable1,thematchwiththe3UTRregionof GBEistoasequence5oftheSEIstartandmaynotbeasourceofCSIROsiRNAs. Again,thisisnotcertaingiventheambiguityofthesourcesequenceforthe siRNAs.However,onesequencematchtothe3UTRregionofGBEisinthe predictedmRNAofSEIinintron13(rowone,Table1).Thismaybeasourceof offtargeteffectsshouldthesiRNAsmigratetothenucleus(Robbetal.,2005). Plausibleriskpathways: FollowingfromtherationalethatsiRNAproducedbygeneticengineering,or secondarydsRNAsthatarecausedbythemodification,maybebiologically relevanttohumanhealthandtheenvironment,Iconsiderpathwaysthrough whichpotentialadverseeffectsmightarise.

10Notethattheywerenotcharacterizedforbeingknownopenreadingframes.
Heinemann:EvaluationofrisksfromcreationofnovelRNAmoleculesingeneticallyengineeredwheat plantsandrecommendationsforriskassessment

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 ProductionofintendedsiRNAmoleculesmaybothcauseintendedgenesilencing andhaveofftargeteffects,i.e.,maysilencegenesotherthanthoseintended. Unanticipatedofftargetadverseeffectscanbedifficulttodetect. Offtargeteffectsarecommon,difficulttopredict,butnotinevitable.Theyarise frommatchesbetweentheintendedsiRNAandsequencesinothergenes (Jacksonetal.,2003,Linetal.,2005,Seinenetal.,2011,Semizarovetal.,2003). SomeunintendedeffectsofsiRNAskillcells(Fedorovetal.,2006),butlesserand stillconcerningadverseeffectscanrequiremoresensitivetechniquestoreveal (Zhangetal.,2012a). Offtargeteffectscanalsoarisefromimperfectmatchesbetweentheintended siRNAandsequencesinothergenes,particularlywhenthesematchesareinthe 3UTRofanRNA(Birminghametal.,2006). Inaddition,offtargeteffectscanarisefromeitherperfectorimperfectmatches betweensecondarilyderiveddsRNAsandsequencesinothergenes(Baumetal., 2007,GordonandWaterhouse,2007,Zhangetal.,2012b). Anyofftargeteffectmaycausesilencingineitheranothergeneofwheat,orinan organismconsumingthewheatorexposedinsomeotherway(possiblythrough inhalation,althoughthisexposurepathwayhasnotyetbeentestedtomy knowledge).Anyofftargeteffectthatcausedthegenerationofadditional secondarydsRNAswouldpotentiallycreateevenmoreunanticipatedofftarget effects. Table1.Highlightedmatchesbetween3UTRregionofGBEandpotential siRNAsofSEI. Nucleotiderange Perfectmatch Overallidentity length (%) GBE SEI 2796 24642 11 82 2465 17647 11 100 2632 11339 11 79 2632 11925 11 100 2647 12745 11 100 2688 8819 11 100 2700 11063 15 100 2807 10211 11 81 2813 15854 11 100 2961 11580 11 100 3073 9243 11 76 ScientificstudiesIwouldrecommendtoprecedeafieldtrial: Withtheplausibleriskpathwaysunderstood,whatkindsofscientificstudiesor assurancesshouldhavebeen,inmyopinion,undertakentoaddressconcerned membersofthepublic?
Heinemann:EvaluationofrisksfromcreationofnovelRNAmoleculesingeneticallyengineeredwheat plantsandrecommendationsforriskassessment

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I.

Bioinformaticsstudiestoidentifyanylikelyunintendedtargetsofthe intendedsiRNAsinhumansandspeciesusedasindicatorsofkeyecological functionsorwhichareprotected.Thesestudieswouldhavelookedfor perfectmatchesorsimilarsequencesinthecodingregionandintrons (Seinenetal.,2011),andperfectmatchesinseedregionsof3UTRs,ofRNAs derivedfromwholegenomesequences,whereavailable.

II. Whenawholegenomesequenceofsufficientconfidenceisnotavailablefora species,morespecificlaboratoryexperimentsmightneedtobeconducted. III. ExperimentalverificationoftheintendedsiRNAs(bothsequenceand structure)inwheatanddemonstrationthatsilencingisbyaknowndsRNA mediatedpathway,determininganyroleforeitherorbothcytoplasmic (Figure2)ornuclear(TGS)silencingpathways. IV. Anypotentialofftargeteffectidentifiedthroughthebioinformaticsanalysis shouldeithercausethesiRNAtoberejectedandanothersought,ifpossible, orbefurtherevaluatedbytissueculturestudies(humanoranimalcells),for exampleasdonebyZhangetal(2012b),longterm(e.g.,atleasttwoyear long)feedingstudies(nonhuman),orinhalationstudies(nonhuman) testingforpotentialsilencingofidentifiedunintendedgenes.Animalstudies cannotsubstituteforuseofhumantissueculturestudiesinahumanhealth riskassessment(Burchardetal.,2009). V. ForanysiRNAnotfoundtocauseanadverseeffectonanimals,further testingshouldbeconductedtoexcludetheinplantaproductionofsecondary dsRNAmoleculeswithotherofftargeteffects,especiallybeforeany purposefulpotentialexposuretohumans.Thiscouldbedonethrougha semitargetedqualitativeprofilingofsmallRNAmoleculesusinghigh throughputsequencinginacomparativeassessmentbetweentheGMand conventionalparent(Heinemannetal.,2011),asimilarcomparative profilingexercisefrom(human,animal)tissuecultureseitherexposedornot totheintendedsiRNAs,and/orproperanimal/insectfeedingandinhalation studiesifnotalreadyconductedabove. Conclusions: (1) ThereareextensivesimilaritiesbetweentheplantSEIgeneandthe humanGBEgene.Thebioinformaticsanalysiscannotruleoutunintendedcross reactivitybetweensiRNAs,designedtosilenceSEI,andGBE. (2) ThereareextensivesimilaritiesbetweenSEI(includingitsintrons)and othergenesinthehumangenome.Thebioinformaticanalysiscannotruleout unintendedcrossreactivitybetweensiRNAs,designedtosilenceSEI,andother genes. (3) Inplants,siRNAscanbesystemicallytransmitted.Itwouldnotbe possiblewithoutexperimentalconfirmationtoensuretheabsenceofthesiRNAs intissueotherthanendosperm. (4) AnRNAieffectcanresultinthegenerationofunintendedsecondary siRNAs.ThesemayextendthepotentialforunintendedcrossreactivitywithGBE orotherhumangenes.

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(5) Itwouldnotbepossibletoexcludeunintendedsilencingeffectswithout propergenetictesting.Unintendedactivitiesarespeciesspecific(Burchardetal., 2009),sotestingshouldbeconductedinanimals,butalsoanimalswith establishedpatchesofhumancelltissue,andusingrelevanthumantissueculture cells. Respectfullyyours

Dr.JackA.Heinemann Professor DirectoroftheCentreofIntegratedResearchinBiosafety

References11:

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