Toward an effective strategy in glioblastoma treatment. Part II: RNA interference as a promising way to sensitize glioblastomas to temozolomide.

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Toward an effective strategy in

glioblastoma treatment. Part II: RNA interference as a promising way to

sensitize glioblastomas to temozolomide

Khaled Messaoudi

^1,2

, Anne Clavreul

^1,2

and Fre´de´ric Lagarce

^1,2,3

1LUNAMUniversite´,Angers,France

2InsermU1066,MicroetNanome´dicinesBiomime´tiques,IBS,AngersCedex9,France

3ServicePharmacie,CHUAngers,France

RNA interference (RNAi) is a strategy of gene regulation that has opened up many opportunities for the treatment of cancers, especially glioblastoma multiforme (GBM). This strategy reduced the expression of many proteins involved in the resistance of these tumors to anticancer drugs, particularly to

temozolomide (TMZ). A significant research effort has gone into RNAi delivery and target selection for clinical application of this new discovery in the treatment of GBMs. However, some limitations must be resolved to enhance the safety of RNAi-based therapeutics and to reduce their immune response. In this review, the mechanism of RNAi will be described. Moreover, the opportunities offered by RNAi strategy to reverse the phenotype of these tumor cells as well as prospects and challenges ahead in the RNAi-based therapy will be discussed.

Introduction

Resistance to anticancer drugs is a problem in many cancers, particularlyglioblastomamultiforme(GBM)–themostcommon typeofprimarybrain tumor[1].Theprognosis ofthesetumors remains poor with a median survival of 14.6 months despite receiving many therapies including surgery, radiotherapy and chemotherapy[2].Combiningradiationtherapywithtemozolo- mide(TMZ)iscurrentlythefirst-linetherapyforGBMs.However, the efficiency of TMZ remains limited owing to inherent and acquiredresistanceofglialtumorcells.Themainresistancemech- anismshavebeendetailedinpartIofthisreviewseries.Different strategiesof inhibiting the effect ofthese proteins involvedin resistancetoTMZweretested.However,theeffectsobtainedare ofteninadequateordisappointing.

Development of the RNA interference (RNAi) strategy has openednewperspectivesforthetreatmentofthesemalignancies.

Indeed,theprincipleofRNAiisbasedonthereductionofexpres- sionofatargetmRNAintoprotein.Thisstrategyhasbeenapplied withsuccessinvitroandinvivoindifferentpathologiesincluding cancers, viral infections and metabolic disorders [3]. However, transport and delivery of interfering RNA requires the use of

vectors that must besafe and efficient. In this review, we will initially describe the RNAi strategy mechanism, followed by a description ofdifferent nonviral vectors used forthe transport anddeliveryofinterferingRNA.Finally,applicationofthisstrate- gyinGBMsandthevariouschallengesandobstaclesthatmustbe overcomeforsuccessfulclinicalapplicationwillbediscussed.

RNAi mechanism in mammalian cells

In recentyears,the strategy ofRNAi, the principleof whichis based oninhibitionof proteinsynthesis by targeting a specific mRNA,wasdiscovered and hasopened up newperspectivesin humandiseasetreatment.RNAiisanintrinsicallycellularpathway thatwasdiscoveredin1998[4,5].TwoclassesofsmallRNAshave been identifiedtomediateRNAi,smallinterferingRNA(siRNA) andmicroRNA(miRNA).

ForsiRNA-mediatedRNAi,thecellularprocessbeginswithlong double-strandedRNAs(dsRNAs)cleavedinthecytoplasmbythe enzymeDicertogeneratematuresiRNAsofabout21–23basepairs (bp). The resulting siRNAs are incorporated into RNA-induced silencingcomplex(RISC),whichbecomesactive.Then,theanti- sense strandremainsinthe RISCcomplexandguidestheRNAi enzymaticmachinerywhilethesensestrandiseliminatedfrom the RISC complex. The RISC-containing guide strand binds to

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Correspondingauthor:Lagarce,F. (frederic.lagarce@univ-angers.fr)

1359-6446/ß2015ElsevierLtd.Allrightsreserved.

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complementarymRNA,whichcausesitsdegradationbytheen- zymeargonaute2(Ago-2)[6,7](Fig.1).

ConcerningmiRNA-mediatedRNAi,genesencodingmiRNAsare transcribed into longprimary transcripts (pri-miRNAs) that are cleavedbyRNAseIIIDroshatogenerateprecursormiRNAs(pre- miRNAs)inthenucleus[8,9].Pre-miRNAsaretransportedfromthe nucleus to the cytoplasm by exportin-5 and then processed by RNAseIIIDicertogeneratematuremiRNAsofabout22nucleotides inlength[8].Then,miRNAsareincorporatedintotheRISCcomplex andbindthroughimperfectcomplementaritytothe3⁰untranslated region(UTR)ofitsmRNAtarget,leadingtotranslationalrepression ormRNAdegradation[8,10].AsinglemiRNAmoleculeisprobably capableofregulatingmultiplemRNAs,andconverselyonemRNA canberegulatedbymultiplemiRNAs(Fig.1).

RNAi delivery

TheuseofthesesmallRNAmoleculesaloneastherapeuticsisnot possibleforseveralreasons.Amongtheseistheirnegativecharge whichpreventsthemfromcrossingtheplasmamembranetoreach

theirtargetinthecytoplasm[6,11].Thepresenceofnucleasesin plasmaand inthe cytoplasmarealsolikelyto deterioratethese biopolymers rapidly after injection [12], not to mention the immuneresponsethatcanbetriggeredbytheirpresenceinthe blood[12].Thesereasonsemphasizetheneedforasuitablevector abletodeliverthesesmallRNAmoleculesintothedesiredcells.

Viraland nonviralvectorsweredevelopedto carrysiRNAsto their site of action located in the cytoplasm [13]. The RNAi inducedbyviraldeliverywasdemonstratedusingdifferentviruses particularly adeno-associated virus (AAV), herpessimplex virus (HSV)andlentiviralvectors[8,14].Despitetheirefficiency,viral vectorshavesomelimitationssuchastheir residualpathogenic effect,whichrepresentsapotentialrisktopatients[15].Moreover, a mutagenesis effect was observed in some clinical trials using thesevectors.Thisiswhyalotofresearchhasbeenfocusedonthe development of nonviralvectors. An ideal nonviral vector has certainattributesthatare:(i)tobebiocompatibleandbiodegrad- able; (ii) to be able to protect nucleic acids against nuclease degradationinplasmaand avoidtheirrenalclearance;and (iii)

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Pleasecitethisarticleinpressas:Messaoudi,K.etal.Towardaneffectivestrategyinglioblastomatreatment.PartII:RNAinterferenceasapromisingwaytosensitizeglioblastomasto temozolomide,DrugDiscovToday(2015),http://dx.doi.org/10.1016/j.drudis.2015.02.014

miRNA gene

Drosha

pri-miRNA pre-miRNA

pre-miRNA Long double strand RNA Cytoplasm

Dicer

miRNA duplex siRNA

Sens strand

RISC complex Sens strand

mRNA target AAAAAAA

mRNA degradation Translation inhibition

Exportin 5

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FIGURE1

MechanismofactionofRNAinterference(RNAi)bysilencingRNA(siRNA)ormicroRNA(miRNA)inmammaliancells.

2 www.drugdiscoverytoday.com

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toallowareversiblebindingofnucleicacidsandtotriggertheir releaseatthesiteofcellularaction.

DifferentcarriersofsiRNAs andmiRNAs weredevelopedand describedintheliterature;mostofthemarepositivelychargedto allowattachmentofnucleicacidsthatarenegativelychargedand toenableabetterinteractionwiththecellmembraneandimprove thecellularuptake[16].Amongthesevectors,therearepolymeric nanoparticles,lipid-basedsystemsandinorganicnanoparticles.

Polymericnanoparticles

Cationicpolymerscaninteractwiththenegativelychargedphos- phate groups of nucleic acids, which create complexes called polyplexes. Natural and synthetic polymers are used for RNAi deliveryinmammaliancells[17].

Polyethyleneimine.Polyethyleneimine(PEI)isasyntheticpoly- merconsideredtobethemosteffectivepolymerforsiRNAdeliv- ery.Thehighdensityofpositivechargeprovidedbythispolymer createsstronglinks withnucleicacids,therebyprotectingthem fromenzymaticdegradationbynucleases.Factorssuchasmolec- ular weight (MW) and degree of branching of PEI affect the efficiencyandthetoxicityofthepolymer[18].Indeed,thehigh MWchainsaremoreeffectiveforsiRNAdeliverycomparedwith thelowMWchains.Similarly,branchedPEIismoreefficientfor siRNA binding than the linear form. However, the high MW chainsandbranchedchainsarethemosttoxic[18](Fig.2).

PEI–siRNAcomplexesareinternalizedbyendocytosisinvari- ouscells.Thispathwayiscomposedofvesiclesknownasendo- somes(withaninternalpHofapproximatelypH5)thatmature from early endosomes to late endosomes before fusing with lysosomes, which contain digestive enzymes [19]. The acidic lysosomal environment causes the protonation of the amine groupsofPEIthat,owingtotheprotonspongeeffectasaresult ofits highbufferingcapacity,inducesosmotic swellingofthe endocytosisvesicleuntilvesiclerupture,leadingtothereleaseof polyplexesintothecytoplasm[20].TheabilityofPEItoescape lysozomaldegradationenablesthereleaseof intactsiRNAand miRNAinthecytoplasmandthusprovidesahighefficiencyof genesilencing[19–21].

Chitosan.ChitosanisanotherpolymerusedforRNAidelivery.

Thisnaturalpolysaccharideconsists ofrepeatingD-glucosamine and N-acetyl D-glucosamine units linked by b(1–4) glycosidic

bonds[22].Itisobtainedbydeacetylationofchitinandisconsid- eredtobeanontoxicandbiocompatiblepolymer[23].

Chitosan MW, degree of deacetylation (DDA) and N:P ratio (ratioofthe protonatedamine groupsofchitosanonthephos- phategroupsofthesiRNA)arefactorsthatgreatlyinfluencesiRNA transfectionefficiency[24].Indeed,higherMWchainsareflexible allowingthemtowrapthesiRNAsinstablecomplexesandprotect themfromnucleasedegradation.However,thisstabilitycanbean obstacleto thereleaseofsiRNAsin thecytoplasm.Inaddition, highMWchitosanshoweddrawbackssuchasaggregationandlow solubilityatneutralpH.LowMWchains enablesmore-efficient intracellular release but low complexation [24]. Higher DDA enhanceselectrostaticinteractionwithsiRNAs,whichleadstoa greaterstabilityofthecomplexesformedwithsiRNAs[25](Fig.2).

The excess ofpositivecharges incomparisonwiththe negative charges increasesthestability ofthechitosan–siRNA complexes andincreasesuptakeacrossanioniccellsurface[25].

Dendrimers. Dendrimers are synthetic macromolecules; their structure is composed of a central core, branches of repeating unitsandterminalgroups,whichcaninteractwithchargedmole- culessuchasnucleicacids,imagingagentsandanticancerdrugs [26,27].Inaddition,theinternalhydrophobiccavitiesofdendri- merscanencapsulatehydrophobicanticancerdrugs[28](Fig.2).

Dendrimerswithpositivelychargedsurfacegroupsareusedfor RNAidelivery[29,30].Poly(amidoamine)dendrimers(PAMAM) arethemostimportanttypeinthedendrimerfamily[31].PAMAM possesstertiaryaminefunctionsintheirstructure,whichcanbe protonated inacidic medium and thusconfera protonsponge effect[32],whichallowsareleaseofsiRNAandmiRNAonthecell level whilepreventingdegradation in the endolysosomalcom- partment[31,33].SomestudiesshowedthatPAMAMdendrimers are excellent nonviral vectors for siRNA and miRNA delivery, thereby producing powerful gene silencing in vitro and in vivo [34–36].Despitetheirtransfectionefficiency,cationicandhigher generationdendrimershavethedisadvantageofinteractingwith bloodcomponents,destabilizingcellmembranesandcausingcell lysis[37].

Lipid-basedsystems

Liposomes. Liposomes are efficient vectors for the delivery of drugs and nucleic acids [38]. Liposomal forms of doxorubicin

Liposome Lipid nanocapsules Dendrimers Polymeric nanoparticles

– –

– ––

– – –

– –

–

– –

– – – – –

– –

– – – – –

– –

– – –

– –

+ +

+

–

– –

– – –

– –

– – – – –

–

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FIGURE2

DifferenttypesofnanocarriersfordeliveryofsilencingRNA(siRNA)usedforRNAinterference.NegativelychargedsiRNAinteractwithpositivechargesofthe nanocarriercomponents.

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andamphotericinBobtainedFDAapprovalforthetreatmentof Kaposi’s sarcoma and fungalinfections, respectively[39]. Lipo- somes areformed by unilamelar or multilamelar phospholipid bilayers.Threeclassesofphospholipidsareusedintheformula- tion of liposomes: anionic,cationic and neutral phospholipids [17,39](Fig.2).

Owing to their hydrophilic nature, the nucleic acids can be encapsulatedinthecoreofliposomes.Conversely,cationiclipids arethemostefficientforthetransportofnucleicacids,because they can interact as cationic polymers that create lipoplexes [20,40]. In addition to their ability to encapsulate drugs, the liposomesurfacecanbefunctionalizedtoallow,firstly,anavoid- anceofrecognitionbytheimmunesystem(i.e.pegylatedstealth liposomes)increasingtheplasmahalf-life[41]and,secondly,to targettumorcellsspecificallythroughtheuseofvariousligands suchasspecificantibodies[42].Despitetheirtransfectionefficien- cyofnucleicacids,cationicliposomeshavethedrawbackofbeing toxicbytheir interactionwith negativelychargedcellularcom- ponents(i.e.opsonins,serumproteinandenzymes)resultingin hemolysisandactivationofthecomplementsystem,whichcauses theirrapidelimination[43].

Lipid nanoparticles. Various types of lipid nanoparticles have beendeveloped.Amongthem,thelipidnanocapsules(LNCs)have astructurethatisahybridbetweenpolymericnanocapsulesand liposomes because oftheir oily core,whichis surrounded by a membranemadeoftensioactiveagentssuchasPEGhydroxystea- rate (Solutol¹). LNCs have many advantagessuchas increased safety,highstabilityandthepossibilityoflipophilicdrugencap- sulation (e.g. paclitaxel) [44]. They can be produced without organicsolventsbyaphaseinversionprocessandwithgenerally recognized as safe (GRAS) excipients and are also genetically modifiedorganism(GMO)-free[45].Theconceptofthisprocess uses the specificabilityof somepolyethoxylated surfactants to modify their affinities for water and oil as a function of the temperature[46].Thesenanocapsuleshaveshowntheirefficiency forthedeliveryofnucleicacids.Indeed,siRNAcanbeentrapped intoLNCsafterformationoflipoplexeswithcationiclipids[47].

Another strategy consists of surface modification of LNCs by graftingacationicpolymersuchaschitosantoobtainacationic vectorcapableof fixingnucleic acidsby electrostatic attraction [48](Fig.2).

Inorganicnanoparticles

Anumberofinorganicnanoparticlessuchascarbonnanotubes, magneticnanoparticlesandgoldnanoparticleshavebeendevel- opedforgenetherapy[49].Thesesystemsaredifferentfromone anotherbecauseoftheircompositionandthemethodsofformu- lationused.

Carbonnanotubes.Somestudieshaveshownthatcarbonnano- tubes easily crossthe plasma membraneusing an endocytosis- independent mechanism [49]. Several functionalized carbon nanotubeshavebeendesignedandtestedforsiRNAandmiRNA delivery[50–53].Themosteffectiveformulationsofcarbonnano- tubeswerefunctionalizedwithaminogroups[54].Despitetheir efficiencyinthetransport ofnucleicacids,theirtoxicity profile causedproblemsfortheiruseintheclinic[55,56].

Magneticnanoparticles.Magneticnanoparticlesweredeveloped fortumorimagingand drugdelivery.siRNAdelivery wastested

withdifferentmagneticnanoparticles,suchasironoxidenano- particles,andhasshownpromisingresults[49].Theadvantageof thesenanoparticlesisthepossibilitytodeliversiRNAtothedesired tissuebyapplyinganexternalmagneticfield[57].

Goldnanoparticles.Goldnanoparticlesarealsousedasnonviral vectorsand present several opportunities forRNAi delivery be- causeoftheirinterestingproperties,in particulartheir biocom- patibilityandlowtoxicity[58,59].Theirstabilityenablesavoiding interactionswith serumioniccompoundsinparticularproteins [60].Goldnanoparticlespossessphotothermalpropertiesthatcan beexploitedforcancertherapy[60].Inaddition,theirsurfacecan befunctionalizedwithspecificligandsto alloweasyfixation of smallRNAmoleculesforgenetherapy[60,61].

RNAi delivery system used to sensitize GBM cells to TMZ

StudiesthatusedRNAitosensitizeGBMcellstoTMZaresumma- rizedinTable1.Theseresultsshowthatdifferenttargetsaretobe reachedbysiRNAforthetreatmentofGBMs.Theadministration ofRNAicanbedonedirectlyintothetumororintravenously.The resultsobtainedinthesestudiesareverypromising.Despitethis, onlyafewnonviralvectorsareapprovedbytheFDAandarein clinicaltrials,demonstratingthattherearestillsomebarriersto overcome,particularlyintermsofselectivity,efficacyandtoxicity.

Amongthevectorsthat havereachedclinicaltrials,theRon- del^TMsystemproducedbyCalandoPharmaceuticalswasusedin clinicaltrialCALAA-01indicatedforthetreatmentofcancersand solidtumorsrefractorytoconventionaltherapy.ItusedansiRNA targetingtheM2subunitofribonucleasereductase,akeyenzyme inthesynthesisandreplicationofDNAthatconvertsribonucleo- tidesintodeoxyribonucleotides.Rondel^TMvectorconsists ofal- ternating b-cyclodextrins and a positively charged polymer to allowattachmentofsiRNAbyelectrostaticattraction;theformed nanocomplexeshaveadiameteroflessthan100nm.Eachcyclo- dextrinmoleculecontainsinitshydrophobiccavityamoleculeof admantane,whichiscovalentlyboundtoonemoleculeofpoly- ethyleneglycol(PEG).TheotherendofthePEGmoleculeislinked toatransferrinmoleculethatwillallowthenanocarriertobindto thetransferrinreceptor(TfR)overexpressedintumorcells[62].

Forclinicalapplication,RNAi-basedcancertherapeuticsshould specificallyrecognizecancercellswithoutaffectingnormalcells.

Theselectivityofnanocarrierswasimprovedbytheirconjugation tospecificligandsthatcanbindthetumorcellreceptors.However, toreachtheirtargetincancercells,itisnecessarytoincreasethe circulationtimeoftheseparticles inplasmabyprotectingthem againstacapturebythereticuloendothelialcellsystem(RES).This protectioncanbeefficientlyachievedthroughaPEGcoatingthat alsohasthedrawbackofreducingthetumorcelluptakeofthese particles[49].

Itiscrucialtoimprovevectorsafetybecauseeventhoughmany vectorshaveproventheirefficiencyinthetransportanddelivery ofdrugsand nucleicacidstheyoftencauseastimulationofthe immune response and cytotoxicity that does not allow their clinical use. The safety profile of vectors could be improved throughthe use ofbiocompatible, biodegradable and nontoxic compounds[49].In addition to efficacy, selectivityand safety, vectorsmustbeproducedona largescale toallowtheir usein clinicaltrials.However, theproduction ofnonviralvectorsata

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thisarticleinpressas:Messaoudi,K.etal.Towardaneffectivestrategyinglioblastomatreatment.PartII:RNAinterferenceasapromisingwaytosensitizeglioblastomastoDrugDiscovToday(2015),http://dx.doi.org/10.1016/j.drudis.2015.02.014

TABLE 1

In vitroand preclinicalin vivoRNAi delivery system used to sensitize glioblastomas to temozolomide.

Delivery system RNAi used Targeted gene

In vitromodel Efficacyin vitro Animal model Route Efficacyin vivo Refs

Viral vectors

Lentiviral vector shRNA MGMT LN18 and T98

GBM cell lines

Inhibition of 80% of the MGMT protein and increase of TMZ sensitivity in both cell types

Subcutaneous tumors in nude mice

Intratumoral Tumor size was reduced by 46% in combination with TMZ

[74]

Plasmid Anti-EGFR

shRNA + wild-type PTEN cDNA

EGFR and PTEN

U251 glioma cells

Downregulation of EGFR expression and upregulation of PTEN expression resulted in suppression of cell proliferation, cell cycle and promotion of cell apoptosis

Subcutaneous tumors in nude mice

Intratumoral The growth of the subcutaneous tumor was significantly inhibited

[75]

Plasmid siRNA Mdm2 Not applicable Not applicable Nude mice Intratumoral Suppression of the HepG2

xenografted tumor growth and reduction of 60% of the Mdm2 protein expression

[76]

Nonviral vectors

LipoTrust^TMliposome siRNA MGMT T98G and U251 GBM cell lines

99% of MGMT knockdown Intracranial tumors in NOD/SCID mice

Intratumoral Response to TMZ significantly increased

[77]

ProFection^WMammalian Transfection

System

siRNA Galectin-1 HS683 GBM cells Inhibition of 60% of the galectin-1 and increase of cytotoxic effect of TMZ in these cells

Nude mice Intratumoral Galectin-1 knockdown in orthotopic xenograft-Hs683 enhanced the TMZ effect and increased the survival of mice

[78]

Lipofectamine^TM siRNA Mutant p53 T98G and U138 GBM cells

p53 knockdown led to a fivefold increase in chemosensitivity to TMZ

In vitromodel Not applicable Not applicable [79]

Chitosan transacylated lipid

nanocapsules

siRNA EGFR U87MG GBM cell line Inhibition of 51.9% of EGFR production on U87MG cells and increase of TMZ sensitivity

In vitromodel Not applicable Not applicable [48]

Polyurethane-short-branch polyethylenimine

miRNA-145 Oct4 and Sox2 GBM-CD133+ cells Increase the sensitivity of treated cells to radiation and TMZ

Intracranial tumors in nude mice

Intratumoral Delivery of PU-PEI-miR145 in orthotopic GBM-CD133+

improved survival in combination with radiotherapy and TMZ

[80]

Abbreviations: EGFR, epidermal growth factor receptor; GBM, glioblastoma multiforme; Mdm2, murine double minute 2; MGMT,O⁶-methylguanine DNA methyltransferase; NOD, non-obese diabetic; PEI, polyethyleneimine; PTEN, phosphatase and tensin homolog; PU, polyurethane; SCID, severe combined immunodeficiency; TMZ, temozolomide.

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largescalehasonlybeendemonstratedwithalimitednumberof drug delivery systems, thisimpedes the use ofthese promising formulationsinclinicaltrials[63].

OnelimitationtotheuseofsiRNAintheclinicsistheoff-target effectduetotheimperfectcomplementaritybetweenthesiRNA andthe3⁰UTRofthemRNAtranscripts.Thisleadstotheinhibi- tionofgenesotherthanthetargetgenesthatmighthaveimpor- tantconsequences forcellfunction[64].Toreduceoreliminate this effect, chemical modificationsof the nucleicacids averted complementarity with othermRNAs other than those targeted [64,65]. The short in vivo effect of siRNA (about one week) is anotherlimitationfortheirusethatrequiresrepeatedadministra- tionandcausesamassiveaccumulationofvectorsandsiRNAinto tumorsresponsiblefortoxicity[66].Itisthusnecessarytoobtain themostbiocompatiblevectorstopreventtheirtoxiceffect.

Thesaturationofthecellularenzymaticmachinerybyexoge- nousinterferingRNAisanotherproblemthatcancausedisruption ofcellularfunctionscontrolledbysmallRNAssuchasendogenous miRNA.Indeed,exportin-5isakeyproteinofthemiRNApathway thatcanbesaturatedwithexogenousmiRNA[64,67].Bycontrast, competition is alsopresent forthe incorporation ofinterfering RNA inthe RISC complex.Thiscompetition isproblematic because the endogenous miRNA present incorporation kinetics slowerthanthatofexogenousRNA[68].

For GBM treatment using the intravenous route, an siRNA carrier must cross the blood–brain barrier (BBB) to release the interferingRNA.Thisbarrierprotectsthebrainfromtoxinsand drugs presentedin thecirculation;thisprotectionisensuredin particular by the tight junctions and efflux pumps such as P- glycoprotein (P-gp) that actively remove these molecules from thecirculation,whichexplainsthefailureofsomechemotherapy [69].Althoughthisbarrierseemstobesomewhatdisruptedinthe abnormal vascular networks characterizingGBMs, it frequently remains intactalong theinfiltrating areawherethe pluralityof recurrencestendtooccur[70].

Bycontrast,thelocaladministrationofnanocarriersavoidsthe crossingoftheBBBandallowsdrugdeliverydirectlyintothebrain parenchyma.Themajordrawbackofthistechniqueisapoordiffu- sionofparticlesinthetumor,whichdoesnotallowthedrugtoreach theareaoftherecurrenceofGBMslocatedafewcentimetersfrom the original tumorarea [71].Moreover,particlereflux fromthe tumorareaisoftenencounteredwhichreducestheeffectivenessof treatment.Thisispartlysolvedbythedevelopmentofconvection- enhanced delivery (CED), which allows injecting the particles underapressuregradient formore-homogeneousdistribution in

thetumor[72].Withthistechnique,thedrugconcentrationsin brain tumors can be superior to those obtained with systemic administration.

Inaddition,GBMsareveryheterogeneoustumorscharacterized by the paralleloverexpression ofmultipleproteins that arein- volvedinresistancetoTMZ.Thisimpliesthat,foreffectivetreat- mentbyRNAi,itisnecessarytoadaptthetherapytoeachpatient basedontheirmolecularcharacteristics.Also,GBMresistanceto TMZcanbeinnateoracquired,whichcancausearapidrecurrence oftumorafterinitialremission.Thisillustratesthatthesuccessofa therapyrequiresknowledgeofthegeneexpressionprofileofthe tumoratdifferenttimestoadaptthetreatmentwithsiRNAand possiblytargetotherproteinsinvolvedinthisnewinducedresis- tance [70]. Despite its effectiveness, drug administration using CEDhas several disadvantages including poordistribution dis- tanceofthedrugtothesiteofplacementofthecatheter(<3cm) whichisaproblemknowingtheinfiltratingandinvasivecharacter ofGBMs [73]. The complexity ofbrain tumors associated with edema and leakage of drugs into the subarachnoid space are drawbacksthatwillhavetobeaddressedfora betteruseofthis technique[73].

Concluding remarks

ConcomitantadministrationofTMZandradiotherapyimproved prognosisofpatientswithGBMbyincreasingmediansurvival,but thiseffectremainsmodest.Thedevelopmentofmolecularbiology hasenabledtheunderstandingoftheinvolvementofcertaingenes in cancer resistance and allowed the molecular profile of each tumortobedefined.RNAiisoneofthemostpromisingstrategies toovercomecancerresistance.Indeed,thisstrategycanbeapplied toachievedifferenttargetsinvolvedin GBMresistancetoTMZ.

Thesetargetscouldbeidentifiedbygeneticscreeningtofindthe genesthatareoutofcontrol foreveryindividual.This concept knownaspersonalizedmedicineadaptsthetherapeuticstrategy foreachpatient,whichisamajoradvantagegiventheheteroge- neityofGBMs.Inaddition,thedevelopmentofnanocarriersable totransportdrugsandnucleicacidsprovidesasynergisticeffect withlowereffectivedosesofthesedrugs and fewerside-effects.

Furtherinvestigationsarestillbeingcarried outto improvethe safety of these nanocarriers and their effectiveness for clinical application.

Acknowledgments

TheauthorsareverygratefultotheLiguecontreleCancer,Comite´

duMaineetLoire,whichfoundedthiswork.

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