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pharmaceuticals Review Aptamer-iRNAs as Therapeutics for Cancer Treatment MarioM.Soldevilla1,2 ,DanielMeraviglia-CrivellideCaso1,2 ,AshwathiP.Menon1,2 andFernandoPastor1,2,* 1 MolecularTherapyProgram,AptamerCore,CenterfortheAppliedMedicalResearch(CIMA), UniversityofNavarra(UNAV),31008Pamplona,Spain;[email protected](M.M.S.); [email protected](D.M.-C.d.C.);[email protected](A.P.M.) 2 NavarreHealthResearchInstitute(IdiSNA),31008Pamplona,Spain * Correspondence:[email protected];Tel.:+34-948-194700(ext.2011) (cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1) (cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7) Received:28August2018;Accepted:10October2018;Published:18October2018 Abstract: Aptamersaresingle-strandedoligonucleotides(ssDNAorssRNA)thatbindandrecognize theirtargetswithhighaffinityandspecificityduetotheircomplextertiarystructure. Aptamersare selectedbyamethodcalledSELEX(SystematicEvolutionofLigandsbyEXponentialenrichment). Thismethodhasallowedtheselectionofaptamerstodifferenttypesofmolecules. Sincethen,many aptamershavebeendescribedforthepotentialtreatmentofseveraldiseasesincludingcancer. Ithas beendescribedoverthelastfewyearsthataptamersrepresentaveryusefultoolastherapeutics, especially for cancer therapy. Aptamers, thanks to their intrinsic oligonucleotide nature, present inherentadvantagesoverothermolecules,suchascell-basedproducts. Owingtotheirhighertissue penetrability,saferprofile,andtargetingcapacity,aptamersarelikelytobecomeanovelplatformfor thedeliveryofmanydifferenttypesoftherapeuticcargos. Herewefocusthereviewoninterfering RNAs(iRNAs)asaptamer-basedtargetingdeliveredagents. Wehavegatheredthemostreliable informationonaptamersastargetingandcarrieragentsforthespecificdeliveryofsiRNAs,shRNA, microRNAs,andantisenseoligonucleotides(ASOs)publishedinthelastfewyearsinthecontextof cancertherapy. Keywords:aptamer;cancer;iRNA;siRNA;shRNA;microRNA;antagomirs;antisenseoligonucleotides; DNA;RNA;therapeutics 1. Introduction RNAorDNAoligonucleotidesarecomposedofnitrogenbaseslinkedbyphosphodiesterbonds. Nucleicacidscandisplayencoding,enzymatic,andstructuralfunctions,duetowhichribonucleotides havebeenestablishedasthekeymoleculesintheoriginoflife[1,2]. Asamatteroffact,thereisawide varietyoffunctionalRNAsinthecell: mRNA,whichencodesgeneinformationtobefurtherprocessed intoproteins;viralRNA,whichcorrespondstotheencodinggenomeofalargefamilyofvirusestermed “RNAviruses”;longnon-codingRNA(lncRNA),whichdonotencodeforanyproteinbutcandisplay scaffoldingstructuralfunctions;interferenceRNA(iRNA),whichregulatesmRNAtranslationand comprisessmallinterferingRNAs(siRNAs),shorthairpinRNAs(shRNAs),microRNAs,antagomiRs (alsocalledantimiRs),andantisenseoligonucleotides(ASOs); structuralRNAs,suchasribosomal RNA(rRNA),transferenceRNAs(tRNAs);andcatalyticRNAswithenzymaticactivity(ribozymes)[3]. ThesedifferenttypesofRNAexerttheirrespectivefunctionsbytwodifferentmodesofaction:sequence specific recognition and structural recognition. Structural recognition of nucleic acids is based on pocketinteractionsimplyingvanderWaalsforces,ionic,hydrogen,andothernon-covalentbonds. Withthebroadcomplexitiesofstructuresandfunctionofnucleicacidsthataredisplayedinnature, itiseasytoenvisionthattheycanbeutilizedaspowerfultherapeuticdrugsfordifferentpurposes[3]. Pharmaceuticals2018,11,108;doi:10.3390/ph11040108 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals2018,11,108 2of22 Sequence specific recognition is mediated by a base-pair fashion, coupling complementary sequencestoexertitsactivatingorinhibitoryfunction. iRNAsworkbysequencespecificrecognition, base-pairingcompletelyorincompletely(dependingontheiRNAsubtype)toitstargetsequenceto modulatemRNAtranslation. RNAitechnologyisbasedinposttranscriptionalgenesilencing(PTGS). PTGSreducestargetleveltranscriptsbysequencespecificrecognitionleadingtolossoffunction[4]. ThefirstevidenceofiRNAactivityinthecellsoccurredserendipitouslyin1990byNapolietal. when overexpression of a specific gene in plants (petunia) did not result in the predicted phenotype [5]. This fact led to the discovery of an intrinsic mechanism of defense against potential foreign RNA, providingthefirstevidenceofiRNAexistence. Thisearlydiscoverytriggeredthedevelopmentof iRNAtherapeutics,whichiscurrentlybasedonsiRNAs,shRNAs,microRNAs,ASOs,etc. iRNAshavebeenusedtomodulatemRNAtranslationineukaryoticcells[6]. Overtime,iRNA technologyhasbeenusedtosilencedisease-causinggeneswhenothermedicinesfailed[6–8]. They currently provide a powerful tool in cancer therapy by regulating tumor-promoting genes [3,8,9]. Nevertheless,despitetheirrelativesuccess,iRNA-basedtherapieshaveencounteredseveralproblems inreachingtheirfullpotentialandkeepongoingforwardasanticancertherapeutics. Biodistribution, stability,off-targeteffectsduetotheirlackofspecificity,andlowefficacyattrespassingcellmembranes arethemajorissueslimitingiRNA-basedtherapiesincancertreatment. Duetotheirprovensuccessas deliveryandtargetingagents,aptamerspresentapromisingtooltodeliveriRNAstothedesiredtarget tissue, increasing their efficacy as anticancer agents. Here we focus this review on aptamer-iRNA chimerasforcancertreatment. 2. Aptamers Aptamers are synthetic single-stranded oligonucleotides (DNA or RNA) of short length (20–100nucleotides)whosethree-dimensionaldispositionconfersthemhighavidityfortheirtargets. Intheearly1990s,theword“aptamer”wascoinedforthefirsttimebyAndrewEllingtonandJack Szostak. They combined the Latin word “aptus” that means “to fit” and the Greek word “meros” thatmeans“particle”tonamethoseselectedRNAspeciesthatspecificallyrecognizedsmallorganic dyes[10]. AtthesametimeLarryGoldandCraigTuerkinparallelselectedspecificRNAligandsfor theT4DNApolymerase[11]. Theselectedaptamersfromthebeginninghaveshownaffinitiesand specificitiescomparable,orevensuperiorinsomecases,tothoseoftheircorrespondingantibodies[12]. Forexample,anaptamerthatrecognizesinterleukin(IL)-6withadissociationconstant(K )of0.2nM d has been recently described [13]. Aptamers are gaining importance as therapeutics due to their potentialadvantagesovercell-basedproducts,suchasrecombinantproteinsorantibodies. Theycan bechemicallysynthesized, whichenablesquickproductionsandversatilechemicalmodifications. Highstability, lackofimmunogenicity, flexiblestructure, andsmallsizeincreasetheirpenetration strength[14]. Oneofthemajorconcernsregardingoligonucleotide-basedtherapiesisplasmastability andtheirhalf-lifeinserum.Aptamerscanbeeasilymodifiedduetotheirchemicalnatureandtherefore optimizetheiryield. Further,custom-tailoredpropertiesofinterestcanbeadded. Specificsubstitutions of OH residues at 2(cid:48) position of ribose for O-methyl or F groups significantly enhance stability in plasma [14,15]. In order to increase the strength of the bond generated between the aptamer and itstarget,non-naturalaromatichydrophobicmodificationsbasedondeoxyUridine(dU)analogous bases,suchasBenzyl-dU(Bn-dU)andNaphthyl-dU(Nap-dU),canbeadded[16]. Thislatterexample is the case of SOMAmers (Slow-Off rate Modified Aptamers) that present protein-like modified sidechains,whichconferthemthefeatureofexposingfewerhydrogenbonds,fewercharge-charge interactions,andfewertotalpolarcontacts[16]. Furthermore,conjugationofaptamerstocholesterol hasshowntoenhancetheirhalf-lifeandbiologicalactivityinvivoinseveralpre-clinicalstudies[17–19]. Moreover,theadditionofclinicallycompatiblecarrierssuchaspolyethyleneglycol(PEG)prevents aptamerexclusionbyrenalfiltration,whichincreasesthetimetheaptameriscirculatinginsidethe organism[14,20]. Forinstance, aPEGylatedanti-MUC1aptamer-doxorubicinconjugateshoweda 6-fold increase in aptamer retention in circulation when compared to unPEGylated aptamers [21]. Pharmaceuticals2018,11,108 3of22 It is also noteworthy that as opposed to what might occur with antibodies, oligonucleotides are unlikely to trigger a T-cell immune response displaying a reduced antigenicity when compared to cell-based products. In what concerns clinical translation, short oligonucleotides can be easily chemicallysynthesizedonalargescaleingoodmanufacturingpractice(GMP)withreducedcosts as compared to proteins that are cell-based derived therapeutic agents. Furthermore, due to their oligonucleotidicnature, aptamerscanbeeasilyneutralizedwiththeirinherentantidotetoreverse any undesirable effect that may arise [22,23]. Aptamers are chemically malleable molecules that canbeconjugatedwithdifferenttypesoftherapeuticcargosinrelativelystraightforwardchemical reactions[14,24]. Aptamers are selected by a biochemical method called SELEX which stands for Systematic EvolutionofLigandsbyEXponentialenrichment. Thisselectionprocedurebeginswitharandom librarypoolofmanydifferentoligonucleotidespecies(1012–15). Eachspeciesofthelibrarycontainsa randomregionofvariablelength(5–50nucleotides(nt))flankedbytwoconstantregionsatthe3(cid:48) and 5(cid:48) end. Thelibrarywillundergoseveralevolutionaryroundsofselectionbeforethebindingspecies areenrichedanddominatethelibrary. Eachroundofselectionconsistsofthreemainsteps: Binding, wherethelibraryisincubatedwiththetargetinordertogetridoftheunboundspecies;Partition,in whichbinderspeciesareseparatedfromthetarget; andAmplification, wherebindersequencesare amplifiedbyPCR(polymerasechainreaction)andfurthertranscribedinvitroifthedesiredresultis anRNAaptamer. Theamplifiedspecieswillserveasasubstratelibraryforthenextroundofselection. The entire SELEX process used to require from 9 to 15 rounds of selection, which implied several monthsofwork,buthavingattheresearchers’disposalnewtools,suchashigh-throughputsequencing (HTS),drasticallyreducesthenumberofroundsandthereforetheamountofworktobedone[24]. HTSfacilitatestheidentificationofalreadyamplifiedspeciesatearlyselectionroundscounteractingthe intrinsicbiasoftheprocedure[24,25].Overthelastfewyears,HTShasbecomeanindispensabletoolfor aptamerselection(HT-SELEX)[25–29]. Sincethefirstaptamerdescribedin1990,theSELEXprocedure hasevolvedandintroducedvariantstooptimizetheselectiondependingonthepurposeandseveral SELEXmethodshavebeendescribed[14,24].TheselectionmethoddescribedbyGold’sandEllington’s groups can be termed conventional SELEX [10,11]. CE-SELEX (capillary electrophoresis-SELEX) uses capillary electrophoresis to purify the binder species [30]. Microfluidic-SELEX (M-SELEX) combines the conventional selection method with a microfluidic system [31]. The introduction of microfluidictechniqueshasenhancedtheefficiencyofotherSELEXmethodssuchasCE-SELEX[32]. TheCell-SELEXprocedureisperformedwithcells,andcounterselectionmightbecarriedoutwith cellslackingtheexpressionofthetargetonthecellsurface[33]. InvivoSELEXcarriesouttherounds ofselectioninliveanimals[34]. Additionally,Toggle-SELEXisusedtoobtaincross-reactiveaptamers between different animal species [35,36]. L-conformed enantiomer aptamers have been described asSpiegelmers[37]. ThefirstSpiegelmeraptamersweredescribedas D-adenosineand L-arginine L-conformed binding aptamers by Fürste’s group [38,39]. Tailored-SELEX is based on identifying shortaptamersthatexcludetheterminalprimer-bindingsitesaspartoftherecognitiondomainusing bridgeadaptorsthatoverlaptheligatesat3(cid:48) and5(cid:48) ends[40]. SyntheticL-mirror-imagedaptamers providetheadvantageofnotinteractingwithoriginalnucleicacidsandnotbeingmetabolizedby thetargetcellaswellaspresentinghighernuclease-mediateddegradationresistance[41]. Moreover, Spiegelmershaveshownveryweakimmunogenicpotential[42]. ThroughSELEX,aptamerscanbeisolatedagainstmoleculesofalmosteverynature,e.g.,lipids, vitamins,sugars,proteins,orevensmallmolecules[14]. Indeed,hundredsofaptamershavebeen publishedtodateforvariousdifferentpurposes[14,24]. Someofthedescribedaptamersarecurrently undergoingclinicaltrialsforthetreatmentofhomeostasis,metabolicdiseases,cardiovasculardiseases, andcancer[43]. Oneofthemostadvancedaptamersincancertreatmentistheanti-nucleolinaptamer AS1411. This26merG-richDNAaptamerhasbeendescribedtobindthenucleolarproteinnucleolin overexpressedonthesurfaceoftumorcells. Itexertsantitumoractivitybyinducingcytotoxiceffects thatinduceapoptosisanditisnowundergoingphaseIandIIclinicaltrialsforthetreatmentofrenal Pharmaceuticals2018,11,108 4of22 cell carcinoma and other solid tumors as well as for acute myeloid leukemia in combination with Cytarabine(https://Clinicaltrials.gov. NCT00881244,NCT00740441,NCT00512083,NCT01034410). EyetechInc®andPfizer®havedevelopedthefirst-in-classaptamertargetingthevascularendothelial growthfactor(VEGF).ThisaptamerhasbeenapprovedbytheFDA(FoodandDrugAdministration) asMacugenin2004,alsoknownasPegaptanib. ItwasthefirstmoleculeofitskindachievingFDA approvalandnowadaysisusedforthetreatmentofAMD(age-relatedmaculardegeneration)[44]. Macugenisa27nt-longpegylatedRNAaptamerwhichtargetsVEGF isoforminordertoprevent 165 choroidal neovascularization [44]. It is a 2(cid:48)-fluoro pyrimidine and 2(cid:48)-O-methyl purine modified aptamerwithadeoxythymidineterminalcaptoincreaseitsstability[44]. Macugenisintravitreally administered at 0.3 mg/eye once every 6 weeks to treat AMD. Nowadays, Pegaptanib is being assessedinclinicaltrialsforthetreatmentofdifferentdiseases,suchischaemicdiabeticmacularedema (MIDME)inphaseIVoruveitisandchoroidalandirisneovascularizationinpilotorPhaseIstudies, respectively [43]. The majority of therapeutic aptamers described to date has been developed to treatcancer,anddespitethefactthatplentyofthemarestillprovidingpreclinicaldata[24],someof theseselectedaptamersarecurrentlyundergoingclinicaltrials. NOX-A12,aSpiegelmerdeveloped byNoxxonPharma® (Berlin,Germany)isa45nt-longaptamerthatblocksthestromacell-derived factor(SDF-1) oralsoknown asCXCL12, achemokine whichpromotes stemcell migration tothe bonemarrowenablingvasculogenesis,tumorgrowth,andmetastasis[45,46]. ThisL-RNAaptameris linkedtoa40kDaPEGunitatthe3(cid:48) endwithnofurthermodificationsandiscurrentlyundergoing PhaseIIclinicaltrialsforthetreatmentofchroniclymphocyticleukemiaandmultiplemyeloma[43,46]. ThemostusedaptamerforcancertreatmentisknownasAS1411,developedbyAntisomaPLC®and previouslytermedasAGRO100. ThisDNAaptamerrecognizestheexternaldomainofthemembrane nucleolinaffectingdifferentcellularpathwayssuchasNF-κ-BandBcl-2[47,48]. AS1411wasthefirst drugtargetingnucleolinandthefirstaptamerinclinicaltrialsforthetreatmentofdifferenttypesof cancer. ItisnowundergoingphaseIIclinicaltrialsforthetreatmentofmetastaticrenalcellcarcinoma (RCC),non-smallcelllungcancer(NSCLC),andacutemyeloidleukemia(AML).TheuseofAS1411 has shown very encouraging results and has shown to be safe and well tolerated by patients [43]. Oneofthemostimportantissuesofnew-generationdrugsistheirtoxicity,andresultsfromclinical trials suggest that the most advanced aptamers, Macugen and AS1411, exert minimal toxicity in patients[49,50]. Newinformationregardingtheadverseeffectsinducedafterlong-termdosageof PegaptanibiscurrentlybeinggatheredfromaPhaseIVstudyforthetreatmentofsubfovealchoroidal neovascularization(NCT00845273). 3. InterferingRNAs InterferingRNAs(iRNAs)areshortRNAsthatexerttheirbiologicalactivitybyhybridizingwith endogenousmRNA(messengerRNA)leadingtothetranslationalrepressionordegradationofthe mRNAtargetgene. Briefly,asshowninFigure1A,DicerprocesseslongRNAtranscriptsintoshort double-strandedRNAswhichareloadedintotheRNA-inducedsilencingcomplex(RISC)alongwith otherproteins,suchasArgonauteandtransactivationresponseRNA-bindingprotein. Thisduplex iRNAconsistsoftwostrands,thepassenger(sensestrand,S),whichisdiscarded,andtheguidestrand (antisensestrand,AS),whichwillcomplementarilypairwiththetargetmRNAsequencehelpedbythe RISC.iRNAtherapeuticscompriseofthedeliveryofsmallinterferingRNAs(siRNAs),shorthairpin RNAs(shRNAs),microRNAmimics(miRNAs),andantisenseoligonucleotides(ASOs). iRNA-based therapiesprovideafeasiblealternativeoptionwhenotheranticancertherapiesfail.TensofiRNA-based therapiesarecurrentlyundergoingclinicaltrialsfordifferentdiseases,severaloftheminPhaseIII. SomeoftheseiRNA-basedtherapiesinclinicaltrialsareaimedattreatingmultipletypesofcancer[51]. Nevertheless,theapplicationofsuchanticanceriRNA-basedtherapiesrequiresmajorconsideration regardingdelivery,efficacy,andmostly,toxicity. Off-targeteffectsofiRNAsowingtoannealingofthe guidestrandtoanon-targetmRNAorundesirableimmunogenicresponsesmaytrigger,insomecases, toxicity[51]. iRNA-basedtherapiesencounterthemajorchallengeofdeliveryasaresultofevolution Pharmaceuticals2018,11,108 5of22 ofdefensestomaintainexogenousRNAsoutsidecells. DuetotheintrinsicnatureofiRNAs,lipid bilayerssuchastheplasmamembraneprovideveryloworabsentpermeabilitytosuchmolecules, which usually results in poor efficacy of iRNA activity [52]. Even though local administration of several iRNAs has been achieved, targeted inhibition of a given gene by systemic administration remainsadifficulttasktoaccomplish. Timeandresourceshavebeeninvestedtosolvesuchproblems; earlyapproachesbeganbyconjugatingiRNAstocholesterolandhumanserumalbumin(HSA)[53]. Lipidnanoparticles(LNPs)providedafeasiblevehiculizationforiRNAdeliveryasdemonstrated by Zimmermann et al. in 2006 due to the masking effect exerted by LNPs that protects the iRNA fromdegradation[54]. Furthermore,iRNAencapsulationpreventsundesirableresponsesowingto engagementofcellulartoll-likereceptorssuchasTLR-3,-7or-8,orintracellularsensorssuchasretinoic acid inducible gene (RIG-I), among others [52]. Synthetic nanoparticles with polymeric materials, suchaspolyethyleneimine(PEI),haveprovedtoefficientlydelivernucleicacidsbuttheiruseoften resultsinassociatedoff-targeteffects[55]. Othersyntheticnanoformulationshaveachievedefficient iRNAdelivery,bothinvitroandinvivo,reducingtargetmRNAexpressionupto85%inhumancells atarelativelylowconcentration(30nM)[56]. SpecificdeliveryofiRNAstohepatocyteshasbeen alsoaccomplishedbyconjugationtoN-acetylgalactosamineinordertotargetanasialoglycoprotein receptor(ASGPR)presentintheliver. Weeklysubcutaneous(SC)administrationofsiRNA-GalNac conjugateresultedinconstanttargetsilencingforninemonthswithnodetectedadverseeffects[57]. Thus,duetothegreatnecessityforinterferingRNAs(iRNAs)tobetargetedtospecificcellstotreata givendisease,thegenerationofaptamer-iRNAchimerasseemstobeaverypromisingtoolforcancer treatment. Herewefocusthereviewonaptamer-basedtargetingofinterferingRNAs,suchassiRNAs, shRNAs,andASOsasdeliveredcargos. Aptamer-iRNAchimerasaresummarizedinTable1. Table 1. Summary of aptamer-based targeted delivery of interfering RNAs for cancer treatment. NSCLC=Non-smallcelllungcancer. TypeofChimera Aptamer iRNA/Target TypeofMalignancy Model Reference PSMA PLK1andBCL2 Prostatecancer LNCaP [58] PSMA PLK1andBCL2 Prostatecancer 22Rv.1(1.7) [59] Coloncarcinomaand CT26and PSMA SMG-1andUPF2 [60] metastaticmelanoma B16/F10 Aptamer-siRNA Melanomaandbreast 4-1BB mTORC1 B16and4T1 [61] cancer EpCAM PLK1 Breastcancer MDA-MB-468 [62] Metastaticbreast 4-1BB IL-2Rα 4T1 [63] carcinoma Metastaticbreast 4-1BB Smad4 4T1 [64] carcinoma CTLA-4 STAT3 T-cellLymphoma Karpas299 [65] PDGFRβ STAT3 Glioblastoma U87MG [66] HER2and EGFR Breastcancer BT474 [67] HER3 CD40 SMG-1 B-celllymphomas A20 [68] PSMA Bcl-xL Prostatecancer LNCaP [69] Aptamer-shRNA PSMA DNAPK Prostatecancer LNCaP [70] AS1411 Bcl-xL Lungcancer A549 [71] AS1411 Bcl-xL NSCLC A549 [72] Axl let-7g/HMG2 NSCLC A549 [73] Axl miR-212 NSCLC A549 [74] Aptamer-miRNA TRA pre-miR-126/VCAM-1 Breastcancer MCF7 [75] miR-26a c-Kit Breastcancer MDA-MB-231 [76] mimic/BAK1 Axl miR-137 Glioblastoma U87MG [77] Pharmaceuticals2018,11,108 6of22 Pharmaceuticals 2018, 11, x FOR PEER REVIEW 6 of 21 mimic/BTAabKl1e 1.Cont. 31 Axl miR-137 Glioblastoma U87MG [77] TypeofChimera Aptamer iRNA/Target TypeofMalignancy Model Reference PC3/Luc Aptamer-ASO AS1411 Luciferase Prostate cancer [78] Aptamer-ASO AS1411 Luciferase Prostatecancer PC73/05L uc705 [78] AxlAanxld and anatni-tmi-miRi-R22-222a2n adnd AptAamptearm-aenrt-ia-mntiiR-miR GGlliioobbllaassttoommaa U8U78M7MG G [79][ 79] PDGPFDRGβFRβ anatni-tmi-miRiR10 1b0b FiguFriegu1re. 1A. pAtpatmamere-ri-RiRNNAA pprroocceessssiinngg ddeeppeennddiningg oonn ththe etytpyep eofo fapatpamtaemr-eiRr-NiRAN Achimcheirma:e r(aA:) (A) AptAamptearm-seirR-sNiRAN(AA (sAiCsi)C;)(;B ()BA) Aptpatmameerr-s-shhRRNNAA;; ((CC)) AAppttaammeer-rm-micircorRoNRNA,A a,nadn (dD()D A)pAtapmtaerm-aenrt-iasenntsisee nse oligoolniguocnleuoctliedoetid(Ae S(AOS).OA).p Atapmtaemr-eirR-iNRANAch cihmimerearab ibninddsst htheea paptatammeerrr reecceeppttoorr aanndd uuppoonn eennggaaggeemmeenntt the chimtheer ac-hriemceeprat-orreciespetmorb iesd edmebdeidndteoda nineton dano ceyntdoosicsytvoessisic vlee.siTchlee. Tchhiem cehriamreeraa crheeaschthese tchyet ocpytloapsmlasamn din caseasnAd ,iBn ,caansdesC A,,t hBe, daundp leCx, itRheN dAuipslerxe cioRgNnAiz eisd rbeycoDginciezreda nbdy loDaicdeerd ainndt oloDaidceedr ainntdo RDNicAer- iannddu ced RNA-induced silencing complex (RISC) assembled to the target mRNA to be further degraded, and silencingcomplex(RISC)assembledtothetargetmRNAtobefurtherdegraded,andthereforeexerts therefore exerts its therapeutic function. In case D, ASOs may exert their function by five different its therapeutic function. In case D, ASOs may exert their function by five different mechanisms: mechanisms: inducing RNAse H-mediated mRNA degradation, preventing 5’-capping and inducingRNAseH-mediatedmRNAdegradation,preventing5’-cappingand3’-polyadenilationofthe pre-mRNA,directinhibitionofproteintranslation,inhibitionofsplicing,orinductionofendogenous micro-RNAdegradation. Pharmaceuticals2018,11,108 7of22 3.1. TherapeuticApproachesUsingAptamer-iRNA 3.1.1. Aptamer-siRNA The use of siRNA-based therapies is frequently limited by tissue-specific targeting and the conjugation with targeting agents increases their efficacy, lowering the doses which decreases the possibility of side effects, while widening the therapeutic window [24]. Aptamer-siRNA chimeras,thathavebeenalsorecentlycoinedAsiCs,arebecomingaveryusefultoolfortargeting gene-knockdown in cancer therapy [14,24]. The first approach to achieve this end was published byLevyandEllingtonin2006. Theygeneratedaconstructconsistingofastreptavidin-biotin-based conjugatetodeliveranti-laminA/Candanti-GAPDHsiRNAstoprostatecancercells. Theconjugate comprisesastreptavidincoreboundtofourbiotinmoleculescovalentlycoupledtoeitherthesiRNA ortheanti-PSMAaptamerA10. PSMAaptamerA10presentsflour(F)groupssubstitutingtheOH groupsatthe2(cid:48) positionoftheribosetoincreaseitsstabilityinplasma. Themajorityofthetherapeutic RNAaptamersdescribedtodateandreviewedhereshowthesamemodification. Thefinalconstruct wasassembledbyincubatingstreptavidinwithbiotinat1:1ratioofsiRNAandPSMAthatshould renderanaverageoftwosiRNAsandtwoPSMAaptamersperconjugate. Theyperformedinvitro experiments adding the aptamer-siRNA chimera to the media at a final concentration of 45 nM (22.5nM ofthesiRNAand 22.5nMof thePSMAaptamer) ortransfectingPSMA-positiveLNCaP tumorcellsat45nMofthesiRNAaswell. TheresultsrevealedthatAsiC-mediatedinhibitionoftarget genesrenderedgeneexpressionatsimilarlevelstothatobtainedbytransfection,whichdemonstrated the advantage of aptamers as delivery agents [80]. That same year, a new class of such reagents was described by McNamara II generating the PSMA aptamer A10 with siRNAs as a single RNA moleculeannealedtothesiRNAguidestrand. Inthiswork,theyusedthePSMA-bindingaptamer A10 to generate different aptamer-siRNA chimeras to target Plk1 and Bcl2 silencing in prostate PSMA-expressing cancer cells [58]. The use of PSMA-Plk1 (A10-Plk1) and PSMA-Bcl2 (A10-Bcl2) AsiCsresultedinspecificbinding,delivery,andsilencingoftargetgenesinPSMA-expressingcells invitro. TheexperimentsperformedonPSMA-lackingprostatecancercells,suchasPC-3,didnot showanysilencingduetotheabsenceofPSMAonthecellsurface. Inordertofurtherdemonstrate thatthedeliveredgenesilencingwasbeingexertedinanaptamer-mediatedmanner,theymutatedthe A10aptamer(A10mut-Plk1)andnobindingnorsilencingwasdetectedinsuchcontrols. Moreover, intratumoraladministrationofA10-Plk1AsiCinLNCaP-bearingathymicmiceresultedinsignificant tumorregressionwhencomparedtothecorrespondingcontrols,suchasPSMA-negativecells(PC-3) and A10mut-Plk1 [58]. Nevertheless, systemic administration of these new kind of reagents was neededtoobtainmoretranslationalrelevanceandshowtheirclinicalfeasibility. Threeyearslater, thesamegrouppublishedanoptimizedversionoftheA10-Plk1AsiCcapableoftargetinghuman PSMA-positivetumorswhentreatedsystemically[59].TheytruncatedthePSMA-bindingA10aptamer from71to39nt(A10-3.2)withoutimpactingitsaffinityinordertoeaseitschemicalsynthesis.Thisnew A10-3.2-Plk1aptamer-siRNAchimerawasfurthermodifiedtoincreaseitsspecificityandsilencing activity. Tothatend,severalmodificationswereintroduced: thelastnucleotideofthe3’endofthe siRNAduplex,whichcorrespondstoaC,wasmutatedforaUinordertofavortheloadingoftheguide (AS,antisensestrand)silencingstrandintoRISC(wobbleconformation);twouracils(U)wereaddedto the3(cid:48)endofthesiRNAduplex;bothstrandsofthesiRNAduplexwereswappedtoprovide5(cid:48)terminal modificationsofthesilencingstrand(swapconformation)tofacilitateDiceraccessibility;andfinally, theaptamer-siRNAchimerawasintegratedintoacontiguouscompletestem-loopsequencetosimulate endogenousmiRNAprecursors(stem-loopconformation). Whilebindingcapacityoftheoptimized AsiCwascomparabletotheoriginalone,theeffectongenesilencingofbothswapandstem-loop conformationwasenhancedupto99%silencingat4nM.Systemicadministrationoftheoptimized AsiCinitsswapconformationinPSMA-expressing22Rv1(1.7)tumor-bearingathymicmiceresultedin asignificantreductionoftumorgrowthandPlk1silencing. Furthermore,PEGylationoftheoptimized swapAsiCsignificantlyincreasedinvivohalf-lifeandinhibitedtumorgrowthatsubstantiallylower Pharmaceuticals2018,11,108 8of22 doses[59].UsingthesameA10PSMAaptamer,Pastoretal.in2010describedanRNAaptamercoupled toasiRNAtotargetNonsense-MediatedRNAdecay(NMD)totumorcellsinordertoenhancetumor antigenicity.NMDisaconstitutivesysteminchargeofcontrollinganddegradingout-of-frameaberrant transcripts containing premature stop codons (PTCs). In the subcutaneous colon carcinoma CT26 murinemodel,thetargetedinhibitionofgenessmg1orupf2significantlyinhibitedtumorgrowthwhen thetargeteddeliveryofcontrolsiRNAsexertedminimalantitumoreffect. Moreover,theintroduction inthisstrategyofboostingagentssuchas4-1BBco-stimulationfurtheraddedantitumoreffects. Within acontextofmetastaticmelanoma,aPSMAtransducedB16/F10melanomacellwasusedtoassess whetherthisstrategywasabletoreachdisseminatedlesions,andthegrouptreatedwithPSMA-smg1 siRNAexertedamoreprofoundlungmetastasisinhibition[60]. Soldevillaetal. recentlydevelopeda newchimeratotreatCD40-expressingB-celllymphomasbytargetingNMDinhibitionaswell. The chimeraconsistsofaCD40RNAagonistaptamercoupledtoashRNAagainstoneofthemainNMD factors SMG-1. This immunotherapeutic chimera resulted in efficient NMD-targeted inhibition in CD40-expressingB-celllymphomaswhileprovidingoptimalconditionsforcancerimmunotherapy; activationofantigen-presentingcells,enhancementoftumorantigenicity,andbone-marrowaplasia recovery[68].In2012,Gilboa’sgrouppublishedausefulsetofrulestorapidlyscreensiRNAsequences foragivenmRNAtargetsuitableforconjugatingwithaptamers,maintainingitsbiologicalactivity[81]. TheinhibitoryactivityofsiRNAstendstodecreasewhenconjugatedtoaptamers,probablydueto thethermosensitivityofthenon-cleavage-baseddisplacementofthepassengerstrandofthesiRNA duplex. RISCneedstwosteps: physicalassociationoftheproteinswithsiRNAduplexandthemRNA, and activation. Activation implies the displacement of the passenger strand, usually limiting the process. Displacement of the passenger strand can be achieved by cleavage or by a non-cleaving manner.ThecleavagecarriedoutbyArgonaute2(Ago2)wouldnotdependonthemeltingtemperature (T )ofthesiRNAduplexprocess,butwhenthepassengerstrandisremovedinanon-cleavedmanner m byAgo1,3,or4,itmightbethermosensitive[82]. Gilboa’sgroupshowedthatdisplacementofthe passengerstrandwoulddependonthemeltingtemperatureofthesiRNAduplexwheresiRNAswith lowT wouldexertahigherbiologicalactivity. Inthiswork, Berezhnoyetal. demonstratedthat m incorporationof2(cid:48)-fluoromodifiedpyrimidinesaffectsthesilencingactivityofthesiRNAaswellasthe T ofthesiRNAduplex. Aptamer-siRNAconjugationimportantlyaffectsthebiologicalactivityofthe m siRNAwheretheAptamer-AS/S(sense,passengerstrand)configurationresultsmoredetrimentalthan Aptamer-S/ASconfiguration. Asmentionedabove,thewobbleconformationoftheAsiCachieved byintroducingamutationonthe5(cid:48) endoftheSstrand(C→U)improvedtheinhibitoryactivityof thechimerawithoutreachingthefulleffectofthenativeunwobbledsiRNA.Moreimportantly,they showed a correlation between the thermal stability and the conjugated siRNA inhibitory activity. TheyalsodemonstratedthatthemajorityofthesiRNAstestedwithacalculatedT lowerthan50◦C m maintained elevated levels of biological activity comparable to those of the native unconjugated siRNAswhensiRNAswithahigherT exhibitlowerbiologicalactivityintheirconjugatedform[81]. m Thesamegroupgenerateda4-1BB-mTORC1AsiCtotargetmTORC1inhibitioninCD8+tumor-specific T lymphocytes. This strategy enhanced their differentiation into CD8+ memory cells leading to a potent antitumor effect in B16 melanoma and 4T1 breast cancer tumor models [61]. Lieberman’s groupdescribedanAsiCconsistingofEpCAMaptamerconjugatedtoPLK1siRNA.This2(cid:48)-fluoro pyrimidine-containingEpCAMaptamerwaslinkedtothesiRNAatthe3(cid:48) endwithdifferentlinkers also adding 3(cid:48)-dTdT overhangs in order enhance its invivo stability, reduce off-target effects, and limitinnateimmunesystemactivation. Thisstrategy,aimedattargetingthedeliveryofPLK1siRNA tobreastcancer,resultedinspecificPLK1knockdowninvitroandinhumanexvivoculturedcells. Theyalsoachievedinhibitionofmammosphereformation,whichimpliesinhibitionofproliferating tumor-initiating cells. In a triple-negative breast cancer MB-468 murine model, the subcutaneous administrationofEpCAM-PLK1siRNAchimerasuppressedtumorgrowth[62]. Recently,Gilboa’s grouphasdescribeda4-1BBaptamerIL-2Ralpha(CD25)siRNAchimeraforthetreatmentofbreast cancer. This AsiC was systemically administered in flank-sited 4T1-bearing balb/c mice resulting Pharmaceuticals2018,11,108 9of22 inapotentantitumorresponsethatpotentiatedtheeffectofirradiatedB7-1andMHC-II-expressing 4T1cellsasvaccines. Thisstrategysignificantlyreducedtumorgrowthaswellasdownregulationof IL-2signalingandthepromotionofamemory-likephenotype[63]. Thissamegroup,andfollowing thesamerationale,generateda4-1BBsmad4AsiCtotargetTGFβinhibitionsignalinginCD8+cells. Inthiscase,thesystemicadministrationofsuchAsiCincombinationofeitherradiation(12Gy)or irradiated B7-1 and MHC-II 4T1 cell-based vaccine enhanced their antitumor immunity [64]. It is knownthatSTAT3worksasaregulatorfavoringtumorformationindifferenttumortypesincluding leukemia,colonandrenalcarcinomas,breastcancers,andglioblastomamultiforme(GBM)[83,84]. Herrmann et al. described an AsiC consisting of the previously described aptamer against the cytotoxicT-lymphocyteantigen4(CTLA-4)[85]andaSTAT3siRNA.Theyshowthatbothlocaland systemicadministrationoftheCTLA-4-STAT3AsiCdrasticallyreducedtumorassociatedregulatory Tlymphocytes(Tregs)invarioustumormodels,aswellasapotentinhibitionoftumorgrowthand metastasis[65]. Franciscis’grouphaverecentlypublishedatargetedSTAT3inhibitionstrategyusing theGint4.TPlatelet-derivedgrowthfactorreceptorβ(PDGFRβ)antagonizingaptamerpreviously described by the same group [86]. They use the Gint4.T aptamer as a targeting agent conjugated to a STAT3 siRNA to target STAT3 inhibition to glioblastoma cells [66]. In the work published by Espositoetal.,theGint4.T-siRNASTAT3chimerawasabletodecreasecellviabilityandmigration invitroinbraintumorcelllinesandmoreimportantlyinhibitingtumorgrowth,decreasingthetumor burden,andinhibitingangiogenesisinimmunodeficientathymicU87MGtumor-bearingmice[66]. AnewapproachhasbeenrecentlydescribedconsistingofatripleAsiCforthetreatmentofbreast cancer. ThisnewchimerawasformulatedasanEGFR(endothelialgrowthfactorreceptor)siRNA disposedbetweentwoaptamers,HER2andHER3[67]. ThisAsiCtermedH2EH3arrestedcellcycle andinducedapoptosisinvitroasdeterminedbyupregulatingcleavedcaspase-3andalsop21inBT474 andSKBR3HER2+HER3+ cells. Atthesametime,thistripletargetingstrategyknockeddownthe threegenes(EGFR,HER2, andHER3)ataproteinlevel. UsingBT474-bearingathymicnudemice asxenograftmurinemodelgroupstreatedintratumorallywiththeH2EH3AsiCreducedtumorsize 1.5-foldwhencomparedtotheadministrationofHER2andHER3aptamermixedwithEGFRsiRNA, and7-foldwhencomparedwiththerestofcontrols. Followingthesametypeofexperiment,butin thiscasewiththetripleAsiC,itwasintravenouslyadministeredandthecomparisonwiththemixture controlrosetoa4-foldchange,whilecomparedtotherestofcontrolmaintainedthesamedifference of 7-fold. It is important to note that imaging experiments revealed no detectable toxicity. ELISA experimentsshowednoIFN-gammaproductionoftripleAsiC-treatedhumanperipheralmononuclear cells(PBMCs)indicatingthatitislikelynottoexertanyundesirableinnateimmuneresponse[67]. ThesizeofthedeliveredRNAmightprovideadditionaltroubleshootingatthetimeoftargeting but,asrecentlydemonstratedbyPorcianietal.,theRNApayloaddoesnotaffecttheaptamer-based targeteddeliveryofsuchmolecules[87]. TheyusedWazandC10.36aptamerstospecificallyinvitro deliver both short and large RNAs to canine CLL17 B-cell lymphoma cells and canine CLGL-90 leukemiaTcells. Theyuseafluorescence“broccoli”-basedtrackingsystemtomonitorthestabilityof intracellular RNAs. Broccoli is a variant of the Spinach fluorogenic RNA aptamer which when complexed with a specific small molecule fluorophore emits fluorescence. The structure of the aptamer-fluorophorecomplexhasbeenusedinthiscitedworkasasensortoassessthemaintenance oftheoriginalfoldingstructureandmeasureRNAdegradation. Theydemonstratethattheaptamer nanostructuretheywereusingremainedhighlystableatearlystagesofendocytosis. Theyalsoshowed thataptamer-RNAconstructsremainedstableandfunctionalinsidetheendosomeforseveralhours, whichmightfacilitatealong-lastingcontrolledreleasetothecytosoltofurtherexertitsfunction[87]. Thisworkopensthedoortothedesigningofnewstrategiesthatfavorthetranslocationofagiven cargo(iRNAs,peptides,drugs,etc.) fromtheendosometothecytosol,therebyenhancingtheefficacy ofaptamer-mediatedtargetingapproaches. Pharmaceuticals2018,11,108 10of22 3.1.2. Aptamer-shRNA As it has been mentioned, another class of interfering RNAs is shRNA. shRNAs are short synthetic iRNA molecules with a hairpin loop-turn that silence target gene expression or target mRNA degradation. shRNAs as can be seen in Figure 1B are recognized and processed by Dicer insidethecytoplasmandinassistancewithRISC,Ago,andotherRNA-bindingproteins,theguide strandishybridizedtothecomplementarymRNAmoleculetoitsfurthercleavageanddegradation resultingingeneknockdown. DeliveryofshRNAintocellsisusuallyachievedusingdifferenttypes ofvectors,whichinthemajorityofthecasesimpliesundesirableoff-targetandimmunogenicside effectsfortherapy[88]. Therefore,efficientdeliveryandtargetingofshRNAsmakeaptamerssolid candidatestoaccomplishthisaim. FollowingthelineofsiRNAs,theycanbeconjugatedtoaptamers asdeliveryandtargetingagents[24]. OnepioneerapproachinshRNAdeliveryusingaptamersfor cancertreatmentwasdescribedbyKimetal. in2010studyingthesynergybetweendoxorubicin(DOX) andshRNA-mediatedBcl-xLgeneknockdown. Theygeneratedaptamer-conjugatedpolyplexes(APs) consistingofDOX-intercalatedpolyethyleneglycol(PEG)-engraftedPEIasnanoparticlescontaining cl-xL specific shRNAs. Invitro studies showed a potent Bcl-xL downregulation after 48 h which, in addition to DOX effect, exerted an excellent antitumor effect [69]. Further reading in aptamer targetnanoparticlescanbefoundintheserecentreviews[14,89–91]. AworkpublishedbyNietal. demonstratedthataPSMA-DNAPK(DNA-activatedproteinkinase)shRNAchimeracouldbeused as a therapeutic agent for prostate cancer [70]. DNAPK is a key factor in DNA repair systems, which is strongly needed after ionizing radiation (IR)-induced DNA damage identified by siRNA library screening. In this work, Ni et al. showed that DNAPK PSMA-based targeted inhibition sensitizedLNCaPprostatecancercellstoionizingradiation,asshownininvitroMTSassayswhere theaptamer-shRNAchimeraincreasedcelldeathafterIRcyclesbutnotthecontrols. Theyassessedits efficacyinLNCaPandPC3prostatetumormodelsandfoundthatthecombinationofradiationwiththe aptamer-shRNAchimerasignificantlyincreasedtumorresponse,moreeffectivelyinLNCaPthanPC3 models. LNCaPtumorsreceivingthecombinatorialtherapyneeded10weekstoreachthesametumor volumeasthosetreatedwiththepropercontrols.Furthermore,exvivoimmunohistochemistryimaging showedthatPSMA-expressinghumantissuewassusceptibletoPSMA-DNAPKshRNAtreatment reducingDNAPKexpressionby25%whencomparedtocontroltreatments[70]. In2015,Askarian etal. publishedthetargeteddeliveryofBcl-XLshRNAinanaptamernanoparticle-basedmanner. Theyconjugatedtheanti-nucleolinAS1411DNAAptamertopolyethylenimineandpolyL-Lysine nanoparticles to specifically deliver a Bcl-XL shRNA-encoding plasmid to lung cancer cells. They achievedasignificantdecreaseinBcl-XLexpressionatbothmRNAandproteinlevels. Theinvitro experimentrevealedinductionofapoptosisinnucleolin-expressingA549lungcancercellsbutnotin nucleolin-negativefibroblastcelllineL929[71]. A new strategy has been generated to effectively transfect shRNAs to cancer cells using the anti-nucleolin aptamer AS1411 as a targeting agent. The AS1411 aptamer was covalently conjugatedtopolyamidoamine(PAMAM)throughanamidebondtoincreasetransfectionefficacy and coupled to a shRNA to inhibit Bcl-xL in target cells. The system was evaluated in A549 nucleolin-expressinglungcancercellsusingL929fibroblastsasanegativecontrol. Theresultsshowed thattheAS1411-PAMAM-shRNAconjugateefficientlytargetednucleolin-expressingcells.Thechimera knocked-downtheexpressionofBcl-xLproteinasdemonstratedbywestern-blot(WB)andselectively inducedapoptosisinnucleolin-expressingcellsbutnotincontrolcellsasshownbyanexinVandPI (propidiumiodide)experiments[72]. 3.1.3. Aptamer-microRNA MicroRNAs (miRNAs) are 19 to 24 nt long non-coding RNAs that function as silencers or post-transcriptionalregulatorsofgeneexpression. TheyareencodedbynuclearDNAandexerttheir activitybypairingwithcomplementarysequenceswithintheirmRNAtarget[92]. Insidethenucleus, genesencodingmiRNAs,usuallylocatedwithinintronicregions,aretranscribedbyRNApolymerase

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Klußmann, S.; Nolte, A.; Bald, R.; Erdmann, V.A.; Fürste, J.P. Mirror-image RNA that binds D-Adenosine. Nat. Biotechnol. Ng, E.W.M.; Shima, D.T.; Calias, P.; Cunningham, E.T.; Guyer, D.R.; Adamis, A.P. Pegaptanib, a targeted.
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