The Yin and Yang of nucleic acid-based therapy in the brain

Review

article

The

Yin

and

Yang

of

nucleic

acid-based

therapy

in

the

brain

$

Stefano

Gustincich

a,b,

*

,

Silvia

Zucchelli

b,c

,

Antonello

Mallamaci

b

a_{DepartmentofNeuroscienceandBrainTechnologies,IstitutoItalianodiTecnologia(IIT),Genova,Italy} b

AreaofNeuroscience,SISSA,Trieste,Italy

c

DepartmentofHealthSciences,Universita’delPiemonteOrientale,Novara,Italy

ARTICLE INFO Articlehistory:

Received1December2015

Receivedinrevisedform16November2016 Accepted20November2016 Availableonlinexxx Keywords: Nucleic-acidtherapy Brain Neurodegenerativedisease Non-codingRNA SmallinhibitoryRNA Antisenseoligonucleotide RNAactivation

NMHV SINEUP

ABSTRACT

Thepost-genomicerahasunveiledtheexistenceofalargerepertoryofnon-codingRNAsandrepetitive elementsthatplayafundamentalroleincellularhomeostasisanddysfunction.Thesemayrepresent unprecedentedopportunitiestomodifygeneexpressionattherighttimeinthecorrectspaceinvivo, providinganalmostunlimitedreservoirofnewpotentialpharmacologicalagents.Hijackingtheirmode ofactions,thedruggablegenomecanbeextendedtoregulatoryRNAsandDNAelementsinascalable fashion.

Here,wediscussthestate-of-the–artofnucleicacid-baseddrugstotreatneurodegenerativediseases. Beneficialeffectscanbeobtainedbyinhibiting(Yin)andincreasing(Yang)geneexpression,depending onthediseaseandthedrugtarget.TogetherwiththedescriptionofthecurrentuseofinhibitoryRNAs (smallinhibitoryRNAsandantisenseoligonucleotides)inanimalmodelsandclinicaltrials,wediscuss themolecularbasisandapplicationsofnewclassesofactivatoryRNAsattranscriptional(RNAa)and translational(SINEUP)levels.

©2016TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Thepost-genomiceraofmoleculartherapy ... 00

2. Challengesforthemoleculartherapyofneurodegenerativediseases... 00

3. TheYinandYangofmoleculartherapyforneurodegenerativediseases ... 00

4. TheYinsideofnucleicacid-basedtherapy:technologies ... 00

4.1. Antisenseoligonucleotides(ASOs) ... 00

4.2. RNAinterference(RNAi) ... 00

Abbreviations:3C,chromosomalconformationcapture;AAV,adeno-associatedvirus;AD,Alzheimer'sdisease;Ago2,argonaute2;ALS,amyotrophiclateralsclerosis; AntagoNATs,antagonistofnaturalantisensetranscript;APP,amyloidprecursorprotein;ASOs,antisenseoligonucleotides;Aub,aubergine;BACE1,b-amyloidprecursor proteincleavingenzyme1;BBB,bloodbrainbarrier;BD,bindingdomain;BDNF,brain-derivedneurotrophicfactor;Cas9,CRISPR-associatedprotein9;CCPG1,cellcycle progression1;CCR4,chemokine(C-Cmotif)receptor4;COX2,cyclooxygenase2;CRISPR,clusteredregularlyinterspacedshortpalindromicrepeats;CSDC2,coldshock domaincontainingC2,RNAbinding;CSF,cerebrospinalfluid;DHX9,DEAH(Asp-Glu-Ala-His)boxhelicase9;ED,effectordomain;Emx2,emptyspiracleshomeobox2;eRNA, enhancerRNA;Foxg1,forkheadboxG1;FRDA,friedreich'sataxia;FTD,frontotemporaldementia;FXN,Frataxin;GFP,greenfluorescentprotein;GOI,gene-of-interest;HD, Huntington'sdisease;HTT,huntingtingene;IGF2,insulin-likegrowthfactor2;IL24,interleukin24;IL32,interleukin32;iPSC,inducedpluripotentstemcells;LDLR,low densitylipoproteinreceptor;lincRNAs,longintergenicnon-codingRNAs;LINE,longinterspersednuclearelement;lncRNAs,longnon-codingRNAs;miRNAs,microRNAs; NAT,naturalantisensetranscript;NMHVs,nuclearlocalizationsignal-MS2coatproteinRNAinteractingdomain-;HAepitope,(3x)VP16transactivatingdomain;PABP,poly (A)-bindingprotein;PD,Parkinson'sdisease;piRNAs,piwi-interactingRNAs;bPLA2G4A,phospholipaseA2;polyA,polyadenylated;PR,progesteronereceptor;PSEN1, presenilin1;RAN,repeat-associatednon-ATG;RAPGEF3,rapguaninenucleotideexchangefactor(GEF)3;RIP,RNAimmunoprecipitation;RISCs,RNA-instructedsilencing complexes;RNAa,RNAactivation;RNAi,RNAinterference;S/AS,sense/antisensepairs;saRNA,smallactivatorRNA;SHANK2,SH3andmultipleankyrinrepeatdomains2; shRNA,smallhairpinRNA;SINE,shortinterspersednuclearelement;SINEUP,SINEB2sequencetoUP-regulatetranslation;siRNAs,smallinterferingRNAs;SLC39A,solute carrierfamily39;SMA,spinalmuscularatrophy;SMN,survivalofmotorneuron;SOD1,superoxidedismutase1;SYNGAP1,synapticrasGTPaseactivatingprotein1;TEs, transposableelements;TFs,transcriptionfactors;TGS,transcriptionalgenesilencing;TSS,transcriptionstartsite;UBE3A,ubiquitinproteinligaseE3A;Uchl1,ubiquitin carboxyl-terminalesteraseL1;UTR,untranslatedregion;VEGF,vascularendothelialgrowthfactor;wt,wildtype.

$ _{ProgressinNeurobiology:Review.}

* Correspondingauthorat:IIT,DepartmentofNeuroscienceandBrainTechnologies,ViaMorego30,16163,Genova,Italy. E-mailaddress:stefano.gustincich@iit.it(S.Gustincich).

http://dx.doi.org/10.1016/j.pneurobio.2016.11.001

0301-0082/©2016TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

xxx–xxx

ContentslistsavailableatScienceDirect

Progress

in

Neurobiology

4.3. Anti-miRNAoligonucleotides(orantagoMIRorblockmir) ... 00

5. TheYinsideofnucleicacid-basedtherapy:applicationstoneurodegenerativediseases ... 00

5.1. Alzheimer’sdisease ... 00

5.2. Amyotrophiclateralsclerosis ... 00

5.3. AmyotrophiclateralSclerosis/Frontotemporaldementia ... 00

5.4. Tauopaties ... 00

5.5. Parkinson’sdisease ... 00

5.6. Huntington’sdisease ... 00

5.7. Transthyretinfamilialamyloidpolyneuropathy ... 00

6. TheYangsideofnucleicacid-basedtherapy:technologies ... 00

6.1. ModifiedmRNAtechnology ... 00

6.2. AntagoNATs ... 00

6.3. Non-degradativeASOs ... 00

6.4. RNAactivation(RNAa) ... 00

6.4.1. Historicalandoperationalfeatures ... 00

6.4.2. Molecularmechanisms ... 00

6.4.3. DownregulatingAStranscriptsinchargeofinhibitingsensetranscriptexpression ... 00

6.4.4. Conveyingtranscription-promotingcomplexestochromatin ... 00

6.5. RNA-programmableNMHVtranscriptionfactors ... 00

6.6. SINEUPs ... 00

7. TheYangsideofnucleicacid-basedtherapy:applicationstoneurodegenerativediseases ... 00

7.1. Angelmansyndrome... 00

7.2. Spinalmuscularatrophy ... 00

7.3. Neurothrophicfactortherapy ... 00

7.4. Friedreichataxia ... 00

8. Conclusions ... 00

9. Competingfinancialinterests ... 00

Acknowledgments ... 00

References ... 00

1.Thepost-genomiceraofmoleculartherapy

Inthelastyears,ourunderstandingofthefunctionaloutputof themammaliangenomehasenormouslyincreased.Thishasledto profoundchangesinthecurrentviewofhowacellworksandhow evolution has shaped biological complexity. These discoveries provide new opportunities for translational research with a potentiallygreatimpactonmolecularmedicine.

Theclassicapproachtodrugdiscoveryhasstemmedfromthe concept that genomes contain the information to encode for proteinsandthesearethebuildingblocksoforganisms.Proteome complexityisthewaytostructurecellsandtissueswithdifferent shapesand functions.Drugs are therefore modifiersof protein activitiesinhibitingoractivatingspecificsignalingpathways.

Despitealonglistofsuccessstoriesthathavepositivelyaffected thewellbeingofpatients,this approachhasposedtremendous challengestodrugdiscovery, resultinginstaggeringcosts,poor specificitiesanddifficultiesinaddressingmajorcomplexdiseases. Importantly,ithasnegativelyimpactedtheabilitytoaddressrare diseasesthatpresenthighlyheterogeneousmolecularpro_filesin spite of the small number of patients. Many proteins remain difficulttobetargeted,limitingtherepertoryoftherapeutics.

Thesequence ofthehumangenomehascon_firmedthat less than5%isinvolvedinencodingproteins, leavingtheremaining sequences,theso-calledJunkDNA,withnofunctionandtherefore noroleinpharmacology.

A pioneering workpreviously showed that JunkDNA might indeedbetranscribedunveilingtheexistenceofsmallRNAsand theexpressionofrepetitiveelements(Kleinetal.,1974).However, thefirstevidencethattheseunconventionaltranscriptsmayhave animportant functional role comesfromthe discoveryofRNA interferenceinthelate1990s(Fireetal.,1998).Immediately,itwas evidentthattheseRNAsmayprovideanewclassofnucleic acid-baseddrugstoinhibitgeneexpressioninvivo.Asforthediscovery ofrecombinantmolecularantibodies,theinitialexcitementand the consequent large investments crashed against technical

difficulties that seemed unsolvable. Twodecades later, we are nowwitnessingarenaissanceofinterestinsmallinhibitoryRNAs duetotherefinementofRNAtargetingandtheoptimizationofin vivodelivery.Morethan50RNAmoleculesandtheirderivatives arecurrentlyformulatedasdrugsandbeingtestedinclinicaltrials for a number of disorders acrossdifferent organs. Overcoming initial failures, we have now entered a second phase of RNA therapeutics,wherewecanoptimisticallyexpecttowitnessthe firstrealimpactofRNAmedicineonpatientsandsociety.

Meantime,largegenomicprojectsasthosefromtheENCODE (Derrienetal.,2012)andFANTOMConsortia(Forrestetal.,2014) have increased enormously the description of the molecular constituentsofcellsandhavecontributedtohampertheclassical viewof gene expressionregulation.In addition toa previously underestimatednumberofalternativevariantsforprotein-coding genes,pervasive transcription of themammaliangenome gives rise to a large repertory of non-coding transcripts, whose expressionistightlyregulated inspaceandtime.Theseinclude long non-coding RNAs (lncRNAs), small non-coding RNAs and transcriptsderivedfromTransposableElements(TEs),suchasSINE (shortinterspersednuclearelement)andLINE(longinterspersed nuclearelement)(Faulkneretal.,2009;Fortetal.,2014;Kapranov etal.,2010;Katayamaetal.,2005).Interestingly,thevastmajority of genomic loci are extensively transcribed from both strands wheretwogenesarelocatedinoppositeorientation,givingriseto Sense/Antisensepairs(S/AS)(Derrienetal.,2012;Katayamaetal., 2005).

In this context, it is becoming evident that the major transcriptional output of mammalian cells is represented by lncRNAs. While functional annotation of the genome has previously revealed almost 15,000 independent lncRNA genes (Derrienetal.,2012),themostrecentversionofLNCpediadatabase contains morethan 90,000annotated humanlncRNAs(Volders etal.,2015).Bydefinition,lncRNAsaretranscriptslongerthan250 nucleotides,withfeaturessimilartothoseofprotein-codinggenes but without a functional open reading frame (ORF). lncRNAs

contain a CAP structure and can be polyadenylated, they are typicallycomposedby2–3exonsandmayhaveintrons,whichare removedduringprocessing(Derrienetal.,2012).Themajorityof lncRNAsareenrichedin thenucleus,albeit theycanexerttheir functionsbothinnuclearandcytoplasmiccompartments.Theycan beclassifiedaccordingtotheirgenomicpositionrelativetonearby genes.WhenlncRNAs are distant fromother protein-codingor non-codinggenes, theyarereferred toas long intergenic non-codingRNAs(orlincRNAs).Whentheyentirelyorpartiallyoverlap withothergenes,theyaredefinedasgeniclncRNAs.Inmostcases, theoverlapisbetweenaprotein-codinggeneononefilamentand an antisense lncRNA on the opposite strand. This genomic architecturegivesrisetonaturalASlncRNAs,whichareestimated tocover almostall protein-coding genes,potentially regulating theirexpression(Derrienetal.,2012;Katayamaetal.,2005).

An increasing number of studies are unveiling lncRNA functions. As representative examples, in the nucleus lncRNAs can have enhancer-like activity (Øromet al., 2010), they may control the epigenetic status of the chromatin by recruiting PolycombRepressorComplex(Rinnetal.,2007)andtheycanactas decoyforinhibitingsplicingandmRNAmaturation(Tripathietal., 2010).Inthecytoplasm,lncRNAsweredescribedtoactivatemRNA decay via Alu elements (Gong and Maquat, 2011), to act as “sponges”formiRNAbinding(Cesanaetal.,2011;Tayetal.,2011) ortoenhancetranslationunderstressconditions(Carrierietal., 2012).Irrespective of thespecificlncRNA andits function,it is becomingevidentthat lncRNAsare organizedintoindependent “domains” that are required to bind protein complexes. RNA folding is now believed to provide functional cues to lncRNA domains, as their primary sequences are poorly conserved. In additiontodomainarchitecture,lncRNAscantakeadvantageofthe exquisitefeatureofnucleicacidbasepairingtospecificallyselect andmodulatetargetRNAand/orDNAmolecules.

Therefore,lncRNAsmayworkasflexiblemodularscaffoldsthat combinedomainsengagedforprotein-bindingwiththosetargeted toRNAand/orDNAsequences(GuttmanandRinn,2012).

Withthisextraordinaryamountofdatainhand,alargeeffortis beingdevotedtounderstandthegrammaroflncRNAsstructure/ functionrelationshipand howtheiractivities integrateintothe biologicalscenariosthescientificcommunityhasbeenunveilingin thelasthalfofthecentury.

Importantly,lncRNAs andsmallRNAspresentunprecedented opportunitiestomodifygeneexpressionattherighttimeinthe correctspaceinvivo,providinganalmostunlimitedreservoirof newpotentialpharmacologicalagents.

RNA-based drugs have the advantage of extending the druggablegenomewithhighspecificitytoalltheproteincoding genesas well as toregulatory regions. By taking advantageof commondeliverysystemstospecifictargetorgans,nucleic acid-basedtherapymaybescalableatafairlylowcostwithrelatively commonpharmacodynamicsandpharmacokineticsproperties. 2.Challengesforthemoleculartherapyofneurodegenerative diseases

Itisfairsayingthatdespiteanenormousamountofknowledge accumulatedalongtheyears,thelargemajorityof neurodegener-ativediseasesremainuncurable.Ageneralproblemisthatthesite ofdegenerationisnotaccessiblefordirectstudyduringlife.Thisis especially relevant during clinical trials for new therapeutic treatments since objective measurements of degeneration are scarce.Importantly,neurodegenerativediseasesarecharacterized byalongpre
symptomaticphaseduringwhichdegenerationis occurring but no clinical symptoms are evident. Therefore, potentialpharmacologicaltreatmentsmayactonlyonsurviving cellsthatcannotprovideasufficientsubstratefordiseasereversal.

Furthermore,thereisstillnoobjectivewaytomakeadiagnosisin pre
symptomaticphasesaswellastomonitordisease progres-sion.Insummary,todatenodrugsseemtoblockorevenslowthe neurodegenerativeprocessinanyofthemostsociallychallenging neurodegenerativedisordersasAlzheimer’sDisease(AD), Parkin-son’sDisease(PD)andAmyotrophicLateralSclerosis(ALS).Westill donotknowtheinitialtriggeringeventsofthemajorityofthese sporadic diseases, limiting ourability toidentifya crucial drug target.

In the case of familial neurodegenerative diseases, genetic testing can provide the identification of patients in the pre-symptomaticphase.Importanly,itnamesthetriggeringcauseof the disease and therefore the main validated drugtarget. It is therefore particularly depressing that notreatmentis available evenforthosediseasessuchasHuntington’sDisease(HD),where thecauseispreciselydefined.

Anincreasingnumberofneurodevelopmentaldisorderswith relatively high prevalence in the general population, including autism spectrum disorders (1%), schizophrenia (1%), epilepsy (0.8%) and intellectual disability (2%), have been recently associatedtoDNAcopynumbervariationsand
inselectcases
tohaploinsufficiencyforspecificgenes.Polypeptidesencodedby these genes are extremely heterogenous including molecules linkedtosynapticfunctions(e.g.,NEUREXIN1A,SHANK2,SYNGAP1) aswellaseffectorsfallinginotherontologygroups(e.g.,enzymes involved in chromatin dynamics)(Coe et al., 2012; Devlin and Scherer, 2012; Merikangas et al., 2009; Tam et al., 2009)). Remarkably, animal models often support the etiological link between reduced allele dosage and neuro-cognitive symptoms inferred fromhumangenetics (Ethertonet al.,2009; Jiang and Ehlers, 2013; Nuytens et al., 2013). Gene therapy of neuro-pathogenichaploinsufficienciesisaformidabletask.Insomecases, genescanbetherapeuticallyexpressedatnon-physiologicallevels withouttoxicconsequences,e.g.SMN1inclinicaltrialsforspinal muscular atrophy (https://clinicaltrials.gov/ct2/show/ NCT02122952) and hamartin in mouse models of tuberous sclerosistype1(Prabhakaretal.,2015).Inthemajorityofcases, anaccuratetuningofgeneexpression,closetophysiologicallevels, isstrictlyrequired.ThisisthecaseofFOXG1,whosehemideletion andduplicationleadtoRett-likeandWestsyndromes,respectively (Florianetal.,2012).Giventhecomplexcontrolofgeneexpression, thesomaticdeliveryofanextra-copyofthediseasegenewiththe full repertory of regulatory sequences hardly looks a scalable therapeuticoption.Ontheotherhand,thehomologous recombi-nation-based, CRISPR/Cas9-nuclease-assistedcorrection of gene deletioninvivowouldbeequallydifficulttoimplementforthree concurrentkeyissues:(1)hugesizeofCas9-cDNA;(2)lengthofthe editor DNA, often in the range of megabases; and (3) strong functional prevalence of non-homologous-end-joining (NHEJ) machineryoverhomologousrecombination(HR)inneuronalcells (Oriietal.,2006;Yangetal.,2013).Toovercometheselimitations, smallerCRISPRenzymes,deliverablebybiosafeAAVvectors,have beenrecentlydiscovered(Kleinstiveretal.,2015;Ranetal.,2015). Furthermore,undesiredindelsevokedbyCRISPRnucleasescould bepreventedbytransientinhibitionoftheNHEJmachinery(Chu etal.,2015;Maruyamaetal.,2015).However,theeditorDNAissue isstillunfixed.Therefore,althoughCRISPRenzymesarepromising toolsforaccuraterescueofsubtlegenelesions,thevastmajorityof neurodevelopmental disorders linked to whole-gene hemidele-tionsremainsatpresentuncurable.

For all neurological diseases requiring genome engineering and/orcorrectionofgeneexpressionlevels,thecentralnervous systemposesspecialchallengesfortherapeuticintervention.First, itisisolatedfromthecirculatorysystembytheBloodBrainBarrier (BBB). While essential nutrients can pass, this vascular gate-controlling system prevents most molecules from entering the

nervoussystemfrombloodcirculation.To avoidthis limitation, nucleicacid-and protein-baseddrugscan bedelivered directly intothebrainbyintratechalinjectionintothecerebrospinalfluid (CSF)or byintranasal administration.Interestingly, it hasbeen shownthatproperlyfunctionalizedexosomes(Alvarez-Ervitietal., 2011)and
morerecently
selectedvariantsofrecombinantAAV vectors(Devermanetal.,2016)canpasstheBBBandeffectively support pan-neural nucleic acid delivery upon intravenous administration. Most importantly, the brain is composed by a staggeringcomplexityinneuronalcelltypesandconnectivitythat pose currently insormontable obstacles in delivering the drug speci_fically into the appropriate cell type and in the correct subcellularorsynapticlocation.

3.TheYinandYangofmoleculartherapyforneurodegenerative diseases

ManipulatingRNAexpressioninvivorepresentsanewstrategy forthemoleculartherapyofneurodegenerative diseases.In this context,afirstclassoftherapeuticdrugsshouldpromote down-regulation of gene expression, in conditions of dominant neurodegenerative diseases caused by the expression of a pathological,toxictargetgene.Therapeuticmoleculeshavebeen developedusingtechnologiesbasedonsmallantisense oligonu-cleotides (ASOs) (Gogtay and Sridharan, 2016; Havens and Hastings,2016)andsmallinterferingRNAs(siRNAs)(Bobbinand Rossi,2016;Lorenzeretal.,2015).

Asecondequallychallengingclassofdrugsshouldbebasedon RNA molecules that can increase gene expression in vivo. Augmented levels and/or activity of modifiers of pathogenic pathways maycontribute to reestablish a homeostaticcellular environment. Hijacking the neuroprotective function of neuro-throphinsmay representan attractive strategy topreserve cell viabilityand function in diseased brain. Most importantly, for patientswithhaploinsufficiencies,a therapeuticapproachbased onRNAdrugs torescuethephysiologicalamountsof thetarget proteinwouldinprinciplebecurative.Unfortunately,nomolecules of this type have been found to date leaving these diseases untreatable(vanBokhoven,2011).

This reviewaims atsharing theexcitementover the recent advancementsin thefield of genomics and on their potential applicationindeveloping newnucleic acid-basedtherapies for braindiseases.Forthefirsttimescientistscanuseaplethoraof molecular toolsto manipulate geneexpression invivo both to inhibit(Yin)andincrease(Yang)theexpressionofvalidateddrug targetgenes. Since theability todecrease expression hasbeen withinreachformanyyears,wewillbrieflydescribeinhibitory technologies while focusing on specific examples in animal modelsofneurodegenerativediseasesaswellasoninitialclinical trials data in humans. Importantly, new technologies are now developedtoincreasetheexpression ofselectedgeneswithout permanentlymodifying host genomes. Here we will focus our attentionon the molecular basis of gene-specific activation of transcription (RNAa) and translation (SINEUP). Since these molecular tools are just at their infancy, no clinical data are available.

Weareconciousthatanefficientdeliveryofexogenousnucleic acidsisatthecoreofdrugdevelopment.Forthismatter,werefer thereaderstoreviewsspecificallyaddressingthisimportanttopic (somerepresentativereviewscitedhere)(Gherardinietal.,2014; Gomesetal.,2015;Kanastyetal.,2013;Liuetal.,2013b;Lorenzer etal.,2015;MagenandHornstein,2014;McConnelletal.,2014; Reissmann,2014;Southwelletal.,2012;Zhangetal.,2013).

4.TheYinsideofnucleicacid-basedtherapy:technologies 4.1.Antisenseoligonucleotides(ASOs)

AntisenseoligonucleotidesorASOsareshort,single-stranded syntheticnucleicacids,tipically8–50nucleotideslong,thatbind target RNA molecules by standard Watson–Crick base pairing (Lundinet al.,2015).Uponbinding, ASOsmodulatetarget RNA function. Chemical modifications are required to achieve ASOs stabilityinbiologicalfluidsandtoincreasetheirpotencyinbinding theirmRNAtarget.SeveralvariantsofASOchemistryhavebeen developed aimingat improving nuclease resistance and overall pharmacokineticproperties (Deleaveyand Damha,2012).In all cases,primary nucleotidesequence isenriched withaseriesof chemicalmodificationsthatoverallchangenucleicacid biochemi-cal properties.Chemical modificationscan targetthe phospho-diester bond along the nucleic acid backbone (as in phosphorothioateorPSASOs,thiophosphoramidateand morpho-linoASOs)orthesugarmojetyatthe20-position(asin20-O-methyl or 2OMe and 20-O-methoxy-ethyl or MOE) (Eckstein, 2014; Yamamotoetal.,2011).Insomecases,modificationsatthesugar mojetyareappliedtoaportionoftheASOsequence,whichisthen combinedwithan internalunmodifiedsequence(asin gapmer ASOs).AdditionalvariantsofASOchemistryincludelockednucleic acid molecules, or LNO, where the sugar 20_{-hydroxyl group is}

tetheredwiththe40-carbonatomandtricycle-DNA oligonucleo-tides (Geny et al., 2016), or tc-DNA, where a conformational constrainisinsertedaroundC30-C40andC40-C50bonds(Goyenvalle et al., 2015; Renneberg et al., 2002). Most recently, additional nucleic acid modifications have been introduced to combine chemicaladductsatthebackboneandatthesugars.Oneclassof suchASOsisprovidedbypeptidenucleicacids,orPNAs,inwhich polyamidelinkagesfullysubstitutethesugarphosphatebackbone (Wancewiczet al.,2010).PNAsare resistanttodegradationbut retain base-pairing capabilities showing enhanced affinity. The choiceofchemicalmodificationforASOdesignhasanimpacton theirmechanismofactionandspecificASOchemistryisselected basedonthetherapeutictarget(Gearyetal.,2015).

TherapeuticASOs workmainlythroughtwomechanisms: 1) RNAseH-mediateddegradationoftargetRNA;2)non-degradative stericblockoftargetRNA(BennettandSwayze,2010;Eversetal., 2015).

ASOswithunmodifiedsugarscaneffectivelytriggerRNAse H-mediateddegradationofbase-pairedtargetmRNAs,thusresulting in gene silencing.RNAseH specificallycleaves theRNAmojety whenboundtoASOsinmRNA-DNAheteroduplex.Single-stranded DNAASOsarethenreleasedfromtheduplexandmadeavailable for catalyzing degradation of additional mRNA molecules for a moreefficientsilencing.ThisclassofASOsiscurrentlythemost widelyusedfortherapeuticapplications.Inparticular,MOEASO gapmers targeting mutant SOD1 arethe _first exampleof ASOs testedin humanclinicaltrialtotreatpatientswithALS (Miller etal.,2013).

Chemicalmodi_ficationsat20_{positionofsugarsrenderASOsand}

pairedRNAsresistanttoRNAseH-mediateddegradation.While hampering degradation mechanism, 20 modifications provide enhancedstability,betterpharmacokineticpropertiesand,most importantly, increased binding to target RNA. Non-degradative ASOsactbycreatingasterichindrancethatblockstargetRNAfrom further processing and therefore inhibits its function. In most cases, non-degradativeASOsnegatively regulate mRNA transla-tion,blockingtherecognitionoftargetmRNAbytheribosomesand ultimatelydown-regulatinggeneexpression.

4.2.RNAinterference(RNAi)

It collectively refers to a large array of natural regulatory processes,conserved fromyeasttohumans, whereendogenous molecular machineries, programmable by small-sized RNAs, negativelyregulategeneexpression(BobbinandRossi,2016).

Three types of small RNAs are involved in RNAi, siRNAs, microRNAs(miRNAs)andPiwi-interactingRNAs(piRNAs).siRNAs derive from Dicer-dependent processing of dsRNAs, in turn originatingfromconvergenttranscription,bidirectional transpo-sonreadthroughorunidirectionaltranscriptionofinvertedrepeats (Elbashiretal.,2001;Fireetal.,1998).miRNAsaregeneratedby processing of long primary transcripts (pri-miRNAs), along the classicalDrosha/Dicerpathwayorthe splicing-machinery-medi-atedmirtronpathway(HaandKim,2014;Leeetal.,1993).piRNA, stillpoorlycharacterizedinmammals,areproducedinDrosophila via Zucchini-mediated parsing of long primary transcripts and theirsubsequenttrimming,andmaybeamplifiedthankstoa ping-pongcyclecatalizedbyVasa,Piwi/AubandAgo3(Brenneckeetal., 2007;Vaginetal.,2006).

miRNAsandsiRNAsareloadedontoRNA-instructedsilencing complexes(RISCs),whichprevalentlyactatpost-transcriptional level (Agrawal et al., 2003). RISC-mediated silencing may be achievedbystraighttarget-RNAcleavage.Thisusuallyoccursupon perfectmatchingbetweenthesiRNAguideincludedintheRISC complexanditstarget,andrequirestheendonucleolyticactivityof theAgo2RISCsubunit.Conversely,prevalentlyincaseof miRNA-programmedRISCs,AgosrecruitGW182,whichinturnconveysto RISCothersilencingmachineries.TheinteractionofGW182with poly(A)-binding protein (PABP) may displace PABP, leading to destabilizationofthetargetmRNAanddecreaseofitstranslation. Furthermore,GW182mayrecruitdeadenylasesandthedecapping complexviaaCCR4–NOTcomplexbridge,furtherdestabilizingthe targetmRNAandjeopardizingitstranslation.AsforpiRNAs,they are loaded onto different RISCs, which are imported into the nucleus and act co-transcriptionally. In these cases, target recognition recruits silencing factors and chromatin modifiers increasinglocallevelsofH3K9me3andleadingtotranscriptional silencing(Iwasakietal.,2015).

Endogenous molecular machineries supporting siRNA- and miRNA-RNAihavebeenextensivelyexploitedtoachieveartificial RNAifortherapeuticapplications(BobbinandRossi,2016;Lametal., 2015). While different types ofexogenous RNAs havebeenemployed, immatureRNAprecursorshavebeenoftenshowntooutperform theirsmallRNAderivatives.Amongthebestperformingeffectors thereare29-bplongds-siRNAprecursorsaswellashairpinRNA precursors(smallhairpinRNAsorshRNAs),basedonbackbonesof endogenouspri-miRNAs.Theseeffectorsmaybedeliveredtotarget cellsaspre-mademolecules,withappropriatechemicalmodi fica-tionstostabilizethemandpromotetheirprocessing(Deleaveyand Damha,2012;Lametal.,2015).Alternativelytheymaybedelivered asDNApolII-orDNApolIII-dependent,cDNA-encodingtransgenesto achieveamorepersistenteffect(Lorenzeretal.,2015).Lentiviraland adeno-associated viral vectors are the mostcommonly used to delivershRNAsandmiRNAsintothebrain,astheyprovidestable expressionwhilebeingpoorlyimmunogenic,replication incompe-tent and safe (Keiser et al., 2016). Recently, great therapeutic potentials are held by engineered versions of adeno-associated vectorsthatare capableof crossing thebloodbrain barrierand efficiently deliver transgenes into the brain even aftera single intravenousinjection(Devermanetal.,2016).

4.3.Anti-miRNAoligonucleotides(orantagoMIRorblockmir) miRNAexpressionandfunctionhasbeenprovedalteredina numberofhumandisorders,suchascancer,autoimmunediseases

and neurodegeneration. As a consequence, there has been an increasinginterestindevelopingtherapeuticstrategiestoinhibit miRNAs in disease conditions. Moreover, therapeutic miRNA targetinghastheadvantagethatasinglemiRNA-drugcanaffect manytargetgenessimoultaneuosly,ultimatelyaffectingthewhole disease pathway. Three main strategies have been adopted to achievedown-regulationofmiRNAactivities:expressionofmiRNA sponges, small moleculeinhibitors of miRNAs and anti-miRNA ASOs.In thefirstcase,expressionof longcodingornon-coding transcriptscontainingmultiplecopiesofmiRNAbindingsitescan actasspongesthatcompetewithnaturaltargetsandreducethe actual concentration of active miRNAs. This strategy has been basedonrecentdiscoveriesthatlncRNAs(lincRNAsand pseudo-genes)canactasnaturalmiRNAsponges(Cesanaetal.,2011;Tay etal.,2011).Currently,theuseofmiRNAspongeshasbeenlimited toinvitroapplicationsandsomepre-clinicalmousemodels(Ebert andSharp,2010).CircularRNAs,referredtoascircRNAs,areanovel classofabundant,nuclease-resistantlncRNAsthataregenerated bycovalentlinkof50and30termini(Memczaketal.,2013;Salzman et al.,2012).circRNAscancompetewithprotein-codingmRNAs andlncRNAsandactasspongesformiRNAs(Hansenetal.,2013). As an example, CiRS-7 has been shown to adsorb miR-7, thus releasingitstargetmRNAs,includingParkinson’s disease-associ-ated

a

-synuclein, from inhibition (Hansen et al., 2013; Lukiw, 2013; Salzman, 2016). Furthermore, from compound libraries screenings small molecules have been identified for their inhibition of the expression of specific miRNAs (Young et al., 2010).However,theirtherapeuticuseisyetlimitedbecauseofhigh IC50andpoorspecificity.Finally,widertherapeuticapplicationis comingfromtheuseofASOstospecificallytargetselectedmiRNAs thus blocking their function. Anti-miRNA oligonucleotides, or antagoMIRorblockmir,aresingle-strandedASOsthattightlybind miRNAs and inhibittheiractivityonthetarget mRNA(s).Many antagoMIRs targeting different miRNAs are currently being developedforhepatitisCvirusinfection,livercancer,heartfailure andmetabolicdisordersoftheheart(LiandRana,2014).Someof them have already reachedtheclinicfor phase I/II testing.For neurodegenerative disorders, antagoMIRs have been used in mousemodelsofADandALS(seebelow).

5.TheYinsideofnucleicacid-basedtherapy:applicationsto neurodegenerativediseases

5.1.Alzheimer’sdisease

Alzheimer’sDisease(AD)isthemostcommon neurodegenera-tive disorder,affectingnearly44millionpeopleworldwide and accountingfor60–80%ofallformsofdementias.AD neuropathol-ogyischaracterizedbythedepositionofinsolubleaggregatesof beta-amyloid(A

b

)andtauinextracellularplaquesand intracellu-larneuro_fibrillarytangles,resultinginprogressiveneuronalloss andcorticalatrophy.ADpatientssufferfromprogressivecognitive impairment, gradual loss of memory and learning capabilities, ultimately leadingto degenerationof theoverall qualityof life (Scheltensetal.,2016).Althoughtheprecisemechanismsthatlead to AD are not fully understood, several studies indicate that processingofamyloidprecursorprotein(APP)hasakeyroleinthe disease.

b

-amyloidprecursorproteincleavingenzyme1(BACE1)is involvedinAPPprocessinggivingrisetoA

b

fragments.Increased BACE1 activity has been observed in AD and associated to neurodegeneration and accumulation of A

b

aggregates. Thus, inactivationofBACE1isconsideredapotentialtherapeuticstrategy totreatAD.Tothispurpose,lentiviralvectorswereusedtodeliver BACE1-targetingsiRNAsintohippocampusofAPPtransgenicmice. Uponinjection,siRNAsloweredlevelsofBACE1,reducedamyloid production and rescued neurodegeneration and behavioural

deficitsinAPPmice(Singeretal.,2005).Inamorerecentstudy, BACE1-specific siRNAs were delivered to the mouse brain by systemic injection of modified exosomes, containing neuron-specificRVGpeptidefusedtoLamp2bexosomalprotein( Alvarez-Erviti et al., 2011). BACE1-siRNA loadedexososomes induced a strongdown-regulationofBACE1mRNA(60%)andprotein(62%)in wt mice, thus demonstrating their efficacy as brain delivery system.SyntheticmiRNAswerealsousedtodecreaseBACE1levels andrescuediseasephenotypeinADmousemodeluponsurgical deliverythroughAdeno-associatedviralparticles(Piedrahitaetal., 2015).

BACE1mRNA ispositivelyregulated byanantisenselncRNA, namedBACE1-AS(Faghihietal.,2008).Interestingly,BACE1-ASis upregulated in brain samplesfrom AD patientsthus becaming itselfatargetforsiRNAstrategiesforAD.Indeed,knockdownof eitheroneofBACE1orBACE1-AStranscriptsbyintra-ventricular infusionofmodifiedsiRNAscausedconcordantdownregulationof both transcripts in APP transgenic mice, ultimately leading to reducedA

b

productionandplaquedeposition(Modarresietal., 2011).

InadditiontosiRNA-basedstrategies,ASOsarealsoemployed toreduceexpressionofproteinsinvolvedinADpathogenesis.ASOs targetingacetylcholinesterase,mutatedAPPaswellasPSEN1had shownpromisingeffectsintransgenicADmodels(Chauhanand Siegel,2007;Farretal.,2014;Fiorinietal.,2013).

Finally,antagoMIRstrategiesarealsounderstudyaspotential therapeuticsforAD.ExpressionofseveralmiRNAsisdysregulated in AD brains, resulting in alteration of molecular targets and pathwaysknowntobeassociatedtoADpathology(DiMecoand Pratico,2016).BACE1mRNA,whosequantityiselevatedinAD,isa directtargetofmiR-188-3p,thatconverselyisdown-regulatedin thebrainofADmice.OverexpressionofmiR-188-3pbyintracranial viraldeliveryreducedBACE1levels,decreasedamyloidinclusions and improved synaptic activity and cognitive functions(Zhang etal.,2014).

5.2.Amyotrophiclateralsclerosis

Amyotrophiclateralsclerosis(ALS)isafatalneurodegenerative disordercharacterizedbytheselectivelossofmotorneuronsinthe spinalcordandbrain.ALSpatientssufferfrommusclestiffnessand severemuscleweakness.Theyultimatelydiebyrespiratoryfailure. TheprogressionofALSisrapidandmostpatientssurviveonly3–5 yearsafterdiagnosis.Themostadvancedantisenseapproach to treat ALS is based on ASOs targeting Superoxide Dismutase 1 (SOD1),aproteinmutatedinfamilialcasesofthedisease.Selection of SOD1-specificASOs, optimizationof deliveryand efficacy of treatment were evaluated in a rat model expressing human mutatedSOD1(SODG93A_{).SOD1-ASOswereinjectedintrocerebral} ventriclesandprovedtoreachthespinalcordcirculatingthrough thecerebrospinal_fluid.ASO-treatedmiceshoweddecreaselevels ofSOD1proteinandmRNAthroughoutthebrainandspinalcord. When ASO infusion was performed before disease onset, transgenicratsdisplayedslowerdiseaseprogressionandincreased survival(+37%)(Smithetal.,2006).

Following encouraging pre-clinical data, the first phase-1 clinicaltrialwasfundedtotestsafety,tolerabilityand pharmaco-kinetics of ASO treatment targeting SOD1 upon intrathecal administration in patients with SOD1 mutation (patients with differenttypes of SOD1mutation wereenrolledin this study). Cerebrospinal fluid samples taken immediately after infusion indicatedthepresenceofthedrug.Analysisofspinalcordsamples takenfromoneparticipantwholaterdiedfromALSindicated:i) higherASOlevelsatthesiteofinjectionascomparedtodistant sites;ii)properestimateofdrug persistenceinthebody (peak concentrationat12hafterinfusionandloweredtobasallevelat

24h).Overall,a15%variationinSOD1levelsinthecerebrospinal fluidwasobserved,predictingthathigherdosesofthedrugsare neededtoachieveeffectivedown-regulation(Milleretal.,2013). Additional antisense strategies are also being developed in SODG93A animal models using siRNAs. Several studies have demonstratedeffectivenessofSOD1silencingapproachinmouse modelsofALS,provingreducedSOD1expression,improvedmotor neuronsurvival,and delayedonset ofALSsymptomsin treated animals(Nizzardoetal.,2012).Inmostcases,silencingRNAsare deliveredasshRNAprecursorsusingviralvectors.

Mostrecently,ithasbeenshownthatmiR-155isincreasedin SODG93A_{miceaswellasinthespinalcordofALSpatients,thus} providinganewtherapeutictarget.Indeed,antisense oligonucle-otide therapy devised to inhibit miR-155 (antagoMIR) was developedandtestedinmice.Aftercontinuousintra-ventricular administration, treated mice showed extended survival and prolongueddiseaseduration(38%)(Kovaletal.,2013).Therapeutic potentialofantagoMIRformiR-155inALSwasfurthervalidatedin amorerecentstudy,wheregeneticablationofmiR-155inSODG93A transgenic micerestoredmolecular signaturesin theperiphery (monocytesignature)andinthecentralnervoussystem(microglia signature), as determined by proteomics and transcriptomics (Butovskyetal.,2015).Finally,intra-ventriculardeliveryofLNA formulationofanti-miR-155moleculedelayeddiseaseonsetand extendedsurvivalinSOD1mice(Butovskyetal.,2015).Theexact molecular targets of anti-miR-155 therapy have yet to be identified.

5.3.AmyotrophiclateralSclerosis/Frontotemporaldementia ExpandedhexanucleotiderepeatsinthefirstintronofC9orf72 genewererecentlyidentifiedasthemostcommongeneticcauseof ALS, FrontoTemporal Dementia (FTD) or concomitant ALS/FTD. Althoughtheexactmechanismsbywhichrepeatedelementscause neurodegenerationarecurrentlyunknown,recentstudiessuggest thatneurodegenerationbyC9orf72ismediatedbyRNAtoxicity, lossofC9orf72genefunctionsor,possibly,acombinationofboth. Assupporttothis hypothesis, RNAfociarepresent in ALS/FTD brains.ExpandedRNAis involvedinrepeat-associatednon-ATG (RAN)translationthatproduces aberrantpeptidesordipeptides polymers.Recently,threeindependentstudiesreportedtheuseof antisense oligonucleotides technology to modulate C9orf72 expressionandRNAfoci(Donnellyetal.,2013;Lagier-Tourenne etal.,2013;Sareenetal.,2013).ASOsweredesignedtotargetthe sequencesupstreamordownstreamthetoxicrepeatexpansion.In all cases, a more pronounced down-regulation of C9orf72 was obtainedwithdown-streamtargetingASOs.ASOsweretestedin patients’fibroblast,patients-derivediPScellsand iPS-differentiat-edtomotorneurons.ASOsmediatedtherapywasabletoefficiently target C9orf72 transcript suppressing RNA foci formation and reversinggene expressionalterations observedinpatientscells. ThepositiveeffectonRNAfocicouldbeobtainedwithASOsthat down-regulateoverallC9orf72levels,targetingintronicregions,as well as with those that speci_fically hybridize to transcripts containing the exanucleotide repeat expansion. The use of sense-specificASOscouldnotcorrectRNAsignatureinpatients’ fibroblastsindicatingthatantisense-specificASOswereprobably required for therapeutic effects. Interestingly, siRNA molecules targeting C9orf72 were also tested and surprisingly proved uneffective.

Finally,inLagier-Tourenneetal.(Lagier-Tourenneetal.,2013),a proof of principle study was performed in vivo by delivering C9orf72-specificASOsinthemousebrainwith intracerebroven-tricular stereotaxic injection. Prolonged down-regulation of C9orf72 was observed. Microarrayanalysisrevealed tolerability ofASOinjectioninvivo,withsmallchangesinoverallexpression

profile,long-termreductionofC9orf72mRNA(30–40%less)and noneuropathologicalorbehaviouraldefects.

Severalstudiesareongoingtobetterdefinethegeneticfeatures ofc9orf72-relatedpathology inpatientsinorder topreparefor futureclinicaltrialstargetingthisgene.

5.4.Tauopaties

InAD,progressivesupranuclearpalsyandinanumberofother neurodegenerativediseasescollectivelyknownastauopathies,the macrotubule-associated protein tau becomes hyperphosphory-latedandaggregatestoformintracellularneuro_fibrillarytangles (Iqbal et al., 2016; Wang and Mandelkow, 2016). Antisense oligonucleotidestrategieshavebeendevelopedtodown-regulate tauexpressionandreducetau-associatedseizuresinmice.Inafirst study,injectionoftau-specificASOsdirectlyintothecerebrospinal fluid reduced levelsof endogenous murinetau throughout the entire central nervous system and cerebrospinal fluid. Down-regulationof tauhad no effect onbaseline motor or cognitive behavior,but wasassociatedtoreduced seizuresin chemically-induced seizures model (DeVos et al., 2013). More recently, additionalASOsweredesignedtotargethumantauandtestedin humanneuroblastomacelllinesandintransgenicmiceexpressing humantau.ThestrongesteffectwasachievedwithASOstargeting splicesites, thus mediatingexon skipping and inducing frame-shifts and premature stop codon, with subsequent mRNA degradation (Sud et al., 2014). siRNAs were also employed to silence Tau levels and rescue disease phenotype in vivo in transgenicmiceexpressingmutatedhumantau(Xuetal.,2014). Recently, expression profiling studies have revealed altered quantities of miRNAs in AD and age-related tauopathies (Qiu etal.,2015),implyingtheirroleinpathogenesis.Asaconsequence, therapeuticmodulationofmiRNAsisexpectedtohaveanimpact onpathwaysimplicatedinthedisease.miR-129wasidentifiedto be downregulated in brain tissues from patients with AD and taupathies(Santa-Mariaetal.,2015).Byinvitroexperiments, miR-129wasfoundtobinddirectlythe30UTRoftaumRNA.Invivo, overexpression of miR-129 silenced tau protein and partially abrogatedtautoxiceffectinaDrosophilamodelproducinghuman tau.Inanotherstudy,intra-braindeliveryofmiR-132mimicsvia osmoticpumpsresultedinthereductionoftauandameliorationof long-termmemoryinanADmousemodel(Smithetal.,2015). 5.5.Parkinson’sdisease

Severalgeneticevidencesindicatethat

a

-synucleinexpression affects PD onset, severity and risk. Increased expression of

a

-synuclein provokes a series of events altering neuronal homeostasisandultimatelycelldeath.While

a

-synucleinisprone toaggregationasthecrucialcomponentofLewyBodies,thegene has been found mutated in rare familial cases. Importantly,

a

-synuclein gene triplication(Farrer et al.,2004; Sekineet al., 2010;Singletonetal.,2003)andduplication(Chartier-Harlinetal., 2004;Ibanezet al.,2004;Nishiokaetal.,2009)promote early-onsetPD,provingthatahigherlevelofwildtype

a

-synucleincan leadtopathogenesis(Devineetal.,2011).Inthiscontext,genetic variantsinitspromoterregionareassociatedtosporadicPDcases with lowerexpression being beneficial(Chiba-Falek and Nuss-baum, 2001). Recent reports support the hypothesis that

a

-synucleinalsoplaysacriticalroleinpropagatingthe neurode-generativeprocessviaprion-like mechanisms.Therefore, thera-peutic strategies aiming at inhibiting its cellular function and expressionareunderintensescrutinytoachievedisease amelio-ration.

A largeeffort is undergoingin optimizing siRNA- and ASO-baseddrugstotarget

a

-synucleinmRNA.Widespreaddeliveryinto

the brain was achieved by fusion of rabies virus glycoprotein peptide(RGV)toextra-exosomalN-terminusofLamp2bprotein (Cooperetal.,2014).Innormalmice,systemicadministrationof siRNA-loaded RGV-exosomes decreased

a

-synuclein mRNA and proteinto40–50% ofits endogenouslevelsinSubstantia Nigra, Striatum and cortex. Most importantly, in transgenic mice expressinghumanphospho-mimicS129D

a

-synuclein,treatment with RSV-exosome-siRNAs reduced aggregated and insoluble proteinsinmidbraindopaminergicneurons.

Morerecently,down-regulationof

a

-synucleinexpressionwas obtained using recombinant adeno-associated virus to deliver

a

-synuclein-speci_ficshRNAintheSubstantiaNigrainnormalrats (Zharikov et al., 2015). Under physiological conditions, a 35% reduction ofmRNA and proteinlevelsdidnot causeany motor deficit or neuronal degeneration. However, when rats were subjectedtochemicalmitochondriaintoxication,mimicking PD-like symptoms,AAV-shRNA targeting

a

-synucleinproved tobe neuroprotective, safeguarding dopaminergic neurons in the Substantia Nigra and dopaminergic terminals in the Striatum whilerescuingmotorfunctions.

5.6.Huntington’sdisease

Huntingtondisease(HD)isaninherited,autosomaldominant, fatalneurodegenerativedisordercausedbyaCAGrepeat expan-sioninthefirstexon ofthegeneencoding forhuntingtin(HTT) protein.PathogenicexpansionofCAGsequenceistranslatedinto anelongatedstretchofpolyglutamines(polyQ)attheN-terminal ofHTTprotein,resultingintheproductionofamutantprotein.The mutant protein is toxic and causes neuronal dysfunction and degenerationleadingtomotorsymptoms,cognitivedecline and psychiatricdisturbances (Andrewet al.,1993; TheHuntington’s DiseaseCollaborativeResearchGroup, 1993).SilencingmutantHTT using nucleicacidswould eliminate thebasis ofHD pathology. However, these approaches have raised some concerns about possiblesideeffectsduetothediminishmentofHTTphysiological functions.Indeed,constitutivelossofwtalleleislethalinmouse embryosandreducedexpressionleadstoneurodegenerative-like phenotypes(Dragatsisetal.,2000;Duyaoetal., 1995),whilerecent data seem toindicate that ablationof HTT in adultneurons is nondeleterious(Wangetal.,2016).Extensivebodyofresearchhas demonstrated that wt HTT is neuroprotectiveand plays funda-mentalrolesinseveralcellularpathways(SaudouandHumbert, 2016). As a consequence, in addition to nonallele-selective silencingapproaches,novelstrategiesarebeingdevelopedaimed atallele-specificsuppressionofmutantHTT(BeaudetandMeng, 2016;Keiseretal.,2016).Nucleicacid-basedtherapeuticstrategies, using ASOs and RNAi, hold great promise. Indeed, several preclinicalstudieshavedemonstratedtheutilityofsuchstrategies toimproveHDneuropathologyandsymptoms.Initialstudiesin patients-derivedcells linesindicatedselectiveinhibitionof HTT proteinexpressionbydouble-strandedandsingle-strandedASOs (Huetal.,2009).Inmousemodels,asustainedtherapeuticreversal of HD pathology was obtained by transient repression of HTT synthesis using HTT-targetingASOs (Kordasiewicz etal., 2012). Transient infusion of HTT-ASOs into the cerebrospinal fluid of symptomaticHDmousemodelseffectivelysuppressedHTTmRNA andproteinlevelsandmutantHTTaccumulation,causingdelayed diseaseprogressionandreversalofdiseasephenotype.SimilarASO infusionintononhumanprimatesshowedeffectivereduction of HTTproteininseveral brainregions (Kordasiewiczetal.,2012). Additionalstudiesdescribedselectivedown-regulationofmutant HTT by allele-specific and single-nucleotide polymorphism-specific ASOs (Carrollet al., 2011; Kay et al., 2015; Ostergaard etal.,2013;Southwelletal.,2014).

AnalternativeapproachforsilencingHTTexpressionat post-transcriptional level is the use of siRNAs to trigger the RNAi pathway. Initial studies using RNAi as a therapeutic strategy enabledthereductionofHTTmRNAandproteininvitroincellular models of HD (Chen et al., 2005). Subsequently, RNAi-based approachwasappliedinvivoinpreclinicaltrialforHD (Harper etal.,2005).SinglebilateralinjectionsofAAVdeliveringanti-HTT shRNAwereadministeredintothestriatumofHDtransgenicmice. SignificantreductionsinmutantHTTmRNAlevels(approximately 55%)wereobserved,accompaniedbyareductionofHTTinclusions andrecoveryofmotor deficits.Severalotherpreclinicalstudies followed in the most recent years, mainly focusing on siRNA delivery systems using nanoparticles or cholesterol-conjugated molecules and adeno-associated vectors (Aronin and DiFiglia, 2014; Keiser et al., 2016). Nonallele-specific silencing by RNAi provedtobewell-toleratedresultinginlong-lastingamelioration ofneuropathologicaldeficitsinmice(Boudreauetal.,2009;Drouet etal.,2009;Dufouretal.,2014)andnon-humanprimates(Grondin etal.,2015;McBrideetal.,2011).SelectivesilencingofmutantHTT wasachievedwithsiRNAs(Pfisteretal.,2009)andartificalmiRNAs (Monteys et al., 2015) targeting allele-specific polymorphisms. Allele-selectivesilencingwasalsodemonstratedinpatients’cells andinmousemodelswithsingle-strandedantisense oligonucleo-tides(ss-siRNAs),thatcombinesimplicityofASOtechnologywith theefficiencyofRNAi(Yuetal.,2012).Finally,thedevelopmentof new-generationofbrain-targetingadeno-associatedviralvectors representsapromisingplatformforsilencingtherapyin neurode-generative disorders (Choudhury et al., 2016; Deverman et al., 2016). In mice, a single intravenous injection of modifiedAAV encoding an artificial miRNAwas sufficient to induceeffective silencingofHTTinthebrain(Choudhuryetal.,2016).

Nonallele-specific HTTsilencing approach for HD using ASO technologyhasrecentlyenteredtheclinicwithaphase1/2 trial (sourceClinicalTrial.gov,2015).Togetherwithsafety,itsaimwill includemeasuringthelevelofmutanthuntingtinproteininthe cerebrospinalfluidusinganewlydevelopedassay(Southwelletal., 2015;Wildetal.,2015).

5.7.Transthyretinfamilialamyloidpolyneuropathy

Transthyretinfamilialamyloidpolyneuropathy(TTR-FAP)isan autosomaldominantinheriteddisease,characterizedby progres-sive neuropathy and cardiomyopathy. Like other forms of TTR amyloidosis,TTR-FAPiscausedbymutationsinthegeneencoding fortransthyretin(TTR).Val30Metisthemostcommonmutationin TTR-FAPpatientsandaccountsfor50%ofTTR-FAPcasesworldwide (Gertz et al., 2015; Parman et al., 2016).Mutated TTR hasthe propensitytomisfoldintoinsolubleandpathologicamyloidfibrils. Theseformextracellulardepositsthataccumulateinthe periph-eralandcentralnervoussystemaswellasintheheart,givingrise totheclinicalmanifestations(Sipeetal.,2014).Assuch,reducing thequantitiesofTTRdepositsiscrucialfortherapeutictreatment ofTTR-FAP.A siRNA-baseddrugtargetingTTRand deliveredby meansoflipidnanoparticlesisinclinicaltrialforTTR-FAP.Data fromphaseIIstudydemonstratearobustknockdownofserumTTR proteinwith>80%reductionaftertwodoses.Multipledoseswere generallysafeandwelltolerated(Suhretal.,2015).Arandomized, placebo-controlled, double-blind phase III study (APOLLO, NCT01960348) is currently ongoing for TTR-FAP patients with Val30MettoevaluatethesafetyandefficacyofTTR-targetedsiRNA therapy.AdditionalsiRNAsequencesanddeliverymethodsarestill underdevelopment. Preclinicaltestingindicatethat RNAi-medi-atedknockdown of TTR can efficiently reduce toxic deposition acrossabroadrangeofaffectedtissuesinamousemodelof TTR-FAP(Butleretal.,2016).

6.TheYangsideofnucleicacid-basedtherapy:technologies 6.1.ModifiedmRNAtechnology

ModifiedmRNAtechnology hasrecentlyemergedas anovel optiontogenetherapyapproach.TheuseofDNAand mRNAas geneticsourceoftherapeuticproteinshasstarteddecadesago,but onlytheadventofnovelchemistrytostabilizemRNAmolecules haspromptedtheuseofmRNA-basedtherapytopre-clinicaland clinicalsettings(Sahinetal.,2014).Bydefinition,mRNAhasthe potentialtoproduceproteinsandpeptidesoftherapeuticinterest with easy scalability in the number of targets. mRNA-based therapeuticsdisplay several advantagesover other nucleicacid strategies:i)deliveredmRNAdoesnotneedtoenterthenucleusto befunctional;ii)mRNAdoesnotintegrateintothegenome;iii) productionis relatively simpleand inexpensive.However,since recentyears,therehavebeentwomajor problemsassociatedto mRNAtherapeutics:i)mRNAishighlyunstableincellsandinvivo to achieve sufficient protein expression and ii) mRNA induces strongimmuneresponsesinthehostorganism,thushampering repeated administrations. A variety of formulations have been developedtoprotectnucleicacidsfromextracellulardegradation by RNAses, to facilitate cellular uptake and enable endosomal escapeandeasyreleaseintothecytoplasm.Nanosizedparticlesare typically employed with different biochemical and biophysical properties: positivelychargedlipids, cationic polypetides, poly-mersandmicelles(ChengandLee,2016;Lietal.,2016a;Reichmuth etal.,2016;Uchidaetal.,2013).Overall,lipidnanoparticlesand polyplexnanomicellesarethemostwidelyusedfortherapeutic mRNAdeliveryinvivoandinclinicaltrials(Reichmuthetal.,2016; Sahinetal.,2014).

Chemically-modifiedmRNAsynthesizedinvitroandcombined with naturally occurring modified ribonucleotides has been employed to overcome the aforementioned obstacles and has shown a great promise as therapeutic tool for gene therapy. Expression ofrecombinantproteins afterdeliveryof chemically modifiedmRNAhasbeenshowntobeeffectiveinmiceand non-humanprimatesinmodelsofanemia,congenitallungdiseaseand myocardialinfarction(Karikoetal.,2012;Kormannetal.,2011; Zangietal.,2013).ModifiedmRNAiscurrentlyinclinicaltrailsas cancer immunotherapy and vaccine (Kallen and Thess, 2014). Alternatively,unmodifiedmRNAhasbeensequence-engineeredto displayhigherstabilityandtranslatabilitywhenintroducedinvivo incomplexednanoparticlesforvaccines(Kubleretal.,2015;Petsch etal.,2012;Schneeetal.,2016)andtherapyinthebrain(Linetal., 2016;Nabhanetal.,2016).Recently,unmodifiedmRNAencoding for erythropoietin has been proven effective in large animals (Thessetal.,2015).

6.2.AntagoNATs

InhibitionofnaturalASlncRNAtranscripts(ornaturalantisense transcriptsorNAT)canbeusedasanindirectmeantomodulate endogenous sense mRNA levels. To speci_fically inhibit NATs, Wahlested and collaborators took advantage of the antisense oligonucleotidechemistrytodevelopAntagoNATs.AntagoNATsare ASOsthat inhibita regulatoryantisenselncRNA,thus indirectly modulating sensegeneexpression.AntagoNATsaredesigned to targetS/ASoverlappingregion,thusblockingASfunctionmainly through RNAse H-mediated degradation (Wahlestedt, 2013). DependingonNATfunction,AntagoNATscanbeusedfor down-regulatinggeneexpression,asforBACE1-AS(Faghihietal.,2008; Modarresi et al., 2011), or for up-regulation, as for BDNF-AS (Modarresietal.,2012)andSCNA1(Hsiaoetal.,2016).

6.3.Non-degradativeASOs

The binding capabilities of non-degradative ASOs have also beenemployedforup-regulatinggeneexpression.Mechanistically this can be obtained by blocking mRNA degradation or by increasingmRNA transcription. ASOs designed tobind miRNAs (referredtoasantagoMIRor blockmir)increasemRNA stability interferingwithmiRNA-mediatedmRNAdegradationpathwayand ultimatelyleadingtoenhancedaccumulationofproteinproducts (LiandRana,2014).Up-regulationofgeneexpressioncanalsobe obtained by ASOs designed to bind AS lncRNAs involved in chromatinremodeling. Inthis context,a class ofASlncRNAs is requiredfor suppressing gene expression byrecruiting compo-nentsofthePolycombRepressorComplex 2(PRC2)tothegene locusfromwhichtheyaretranscribed.ASObindingtolncRNAcan thereforeblockPRC2complexassemblyallowingtranscriptionto restart,thusinducingup-regulationofsenseprotein-codingmRNA presentatthesamelocus(Zhaoetal.,2010).

Alternatively,ASOsthatdonotdegradetargetmRNAscanbe designed to modulate their processing. When targeting pre-mRNAs,ASOscanmodulatesplicingandarereferredtoas exon-skipping ASOs or splice-switching oligonucleotides (SSOs) (HavensandHastings,2016).SSOsaredesignedtointerferewith RNA sequences required for the splicing machinery thus inhibitingpre-mRNAinteractionwiththespliceosomeincluding small nuclear RNAs and splicingfactors. Asa result, SSOscan repairdefectivesplicing,promoteexon-skippingorinduce exon-retention, thus restoring the production of a functionally corrected protein. SSOs are usually synthesized using LNA or PNA chemistry to avoid target RNA degradation and achieve potent binding. SSOs have been used in the clinic for the treatmentofDuchenneMuscolarDystrophy(Hodgkinsonetal., 2016;KoleandKrieg,2015;Mendelletal.,2016).Inthecontextof neurodegenerative disorders, therapeutic SSOs have been designedtotargetATMinAtaxiaTelangectasia(Du etal.,2011, 2007), SMN2 in Spinal Muscolar Atrophy (see below) and microtubuleassociatedproteinTauinFrontotemporalDementia and Parkinsonism linkedto chromosome17(FTDP-17) (Peacey etal.,2012).Astate-of-the-artdescriptionoftheuseofASOsas splicingmodifierscanbefoundelsewhere(HavensandHastings, 2016;Koleetal.,2012;Rigoetal.,2014;Sivaetal.,2014). 6.4.RNAactivation(RNAa)

6.4.1.Historicalandoperationalfeatures

Thefirstreportoftranscriptional stimulationbysmallRNAs, brieflyRNAactivation(RNAa),datesbackto2006,whenLietal.(Li et al., 2006) showed that siRNA-like molecules targeting the promoters of three genes of oncological interest, Cadherin E, p21WAF1/CIP1_{andVEGF,wereabletoelicitaspecificandprolonged} stimulation of their transcription in human cells. Since then a plethora of similar reports corroborated these early findings, pointingtoRNAaasapervasivephenomenon(Faghihietal.,2010), that is potentially implicated in endogenous regulation of transcriptionandofgreatinterestfortherapy.Currentlywestill havelimitedunderstandingofitsmolecularmechanisms.Despite theirlikely mechanistic heterogeneity, RNAa processes share a well-definedsetofphenomenologicaltraits.

First, differenttypesof small activating RNAs (saRNAs) may triggerRNAa.TheyincludesiRNA-precursor-like21nt-dsRNAs(Li etal.,2006), pre-miRNA-likeshRNAs(Turunenetal.,2009)and pri-miRNA-like molecules (Diodato et al., 2013). Albeit not necessary, specific covalent modifications may enhance their activity(Kangetal.,2012;Matsuietal.,2010;Placeetal.,2010, 2012).EffectivesaRNAsareusuallydirectedagainstTranscription StartSite(TSS)(Lietal.,2006;Schwartzetal.,2008)orpolyA-site

sequences surroundings (Yue et al., 2010), conserved cis-active modulesregulatinggene-of-interest(GOI)transcription(Diodato et al., 2013)and the transcribed regionof thegene (Liu et al., 2013a).Theymaybebothsense-andantisense-oriented(Diodato etal.,2013).Importantly,finelocationofthesaRNA-targetoftenis akeyissuesinceshiftingitbyonlyafewbasescandramatically affectitsoutcome.MismatchesbetweenthesaRNAanditstarget maybetolerated,howeveronlyiffarfromsaRNA50end(Lietal., 2006).

GeneresponsivitytosmallRNAsdependsincomplexwayson thedifferentiativestateofcellsandonthefunctionalstateofthe targetgene.Insomecases,lowbasalexpressionofthisgenemay predispose to transactivation(Yue et al., 2010). In other cases, RNAaisrestrictedtocellsandtissuesexpressingthetargetgene (Diodato et al., 2013; Li et al., 2006). In these latter cases, generalized delivery of aRNAs might be employed to stimulate selective expression of defective genes within theirexpression domainwithnorisksofectopicactivation.

Whencompared toRNAi, RNAa oftendisplays a later onset, usuallywithadelayof24–48h(Placeetal.,2010),anditlastsquite longer since one unique saRNA delivery may exert an effect prolongedover>7days(Lietal.,2006;Placeetal.,2010).

Finally,mRNA-upregulationachievedviaRNAaisusuallywithin physiologicalrange(often<5-folds)Thisisimportantsincesubtle changesoftargetmRNAlevelsmaydeeplyimpactthefunctional state of thecell, giving riseto predictableand overtbiological effects(Diodatoetal.,2013;Janowskietal.,2007;Voutilaetal., 2012).

Until now, many genes of oncological and cardio-vascular interest have been upregulated by RNAa for purposes of experimental therapy. In this respect, RNAa-mediated gene stimulation is of obvious therapeutic interest for dozens of different hemizygous gene deletions at the basis of a wide spectrum of neuropathological conditions, including epilepsy, mental retardation, autism, schizophrenia and neurodegenera-tion(Laletal.,2015;Leeetal.,2015).Furthermore,upregulation of specific endogenous genes may help counteracting conse-quences of trauma and neurodegeneration (Modarresi et al., 2012).

6.4.2.Molecularmechanisms

We are stillfar fromfull understandingof RNAa dynamics. ExperimentaldatasuggestthatRNAaisaheterogeneousprocess andatleasttwoclassesofmolecularmechanismsmayunderlieit. RNAa may takeplace viapost-transcriptional/co-transcriptional downregulationofAS-transcriptsinchargeoflimitingS-transcript expression. Alternatively, saRNAs may convey supramolecular complexesstimulatingtranscriptiontotargetchromatin. 6.4.3.DownregulatingAStranscriptsinchargeofinhibitingsense transcriptexpression

As described previously, the largemajorityof sensemRNAs presentsantisensetranscription(Katayamaetal.,2005).Insome cases,antisenseRNAscaninhibittranscriptionofitssensegene andsaRNAscanthenfunctionbydownregulatingthe anticorre-lated,antisensecompanionofthetranscripttheypromote.This phenomenonhasbeendocumentedinanumberofexamplesin vitro,(Katayama et al.,2005;Faghihiet al.,2010; Morriset al., 2008;Modarresietal.,2012).Inthesecases,saRNAsmightactby preventing the repressive regulatory effects exerted by the AS transcriptonsensetranscription(Fig.1A).Consistentlywiththis inference,siRNA-mediated, Ago2-dependentknock-downof the convergent p21 AS partner, Bx332409, was followed by the detachmentof the repressiveepigenetic mark H3K27me3from thesurroundingsofthep21-TSSandbysubsequentupregulationof p21-mRNA(Morrisetal.,2008).

6.4.4.Conveyingtranscription-promotingcomplexestochromatin saRNAs may also act by helping to convey macromolecular complexes catalyzing transcription to the gene of interest. Interstingly, saRNA may directly interact with its target DNA (Fig.1B)(Huet al.,2012)or vianascent ncRNAmolecules, still attachedtochromosomal DNA(Fig.1C)(Schwartzet al., 2008; Matsuietal.,2013).

AmongpotentialsupportersofRNAa,Ago1andAgo2interact withmiRNAs andsiRNAandareboth detectableinthenucleus (Huangetal.,2012).Whiletheformerhasascattereddistribution inthenucleoplasm,thelatterisconfinedtotheperipheralregion adjacentthenuclearenvelope (Huang et al.,2013).Ago1 binds pervasively to chromatin, preferentially to TSS surroundings, interactswithRNApolIIandisinvolvedinregulationof transcrip-tion.Assuch,itwassupposedtobespecificallyimplicatedinRNAa. Unexpectedly,however,Ago1isrequiredfortranscriptionalgene silencing(TGS)butnotforRNAa(Chuetal.,2010;Lietal.,2006). ThesameappliestoitsparalogsAgo3andAgo4(Chuetal.,2010). Conversely,ina number ofcasesincludingthose ofp21 and E-Cadherin(Lietal.,2006),PR(Chuetal.,2010),LDLR(Matsuietal., 2010)andCOX2(Matsuietal.,2013),Ago2resultedtobelinkedto targetgenechromatin(orncRNAsstemmingfromit)ina saRNA-dependentmannerandrequiredforRNAaactivity.Inthecaseof COX2-RNAa,Ago2andthesaRNAwereassociatedtoGW182,which wasneededforRNAaaswell(Matsuietal.,2013).Thereasonof selectiveAgo2requirementforRNAaremainsobscure.

BeyondAgo2andGW182,concomitantlywithRNAa,otherkey players are recruited to target gene chromatin or to RNAs stemmingfromit.AnenrichmentofRNApolymeraseII(RNApolII)

atthe50ofgenesupregulatedviaRNAawasdetectedbyChIPorRIP (Huangetal.,2012;Placeetal.,2008;Matsuietal.,2010;Chuetal., 2010;Yueetal.,2010;Majidetal.,2010).ThissuggeststhatsaRNAs oftenactbyeasingtherecruitmentofRNApolIItothetargetgene promoter. Alternatively, miR-483 upregulated IGF2-mRNA, by conveying theRNAhelicase DHX9totheCG-rich50UTRof this mRNA(Liuetal.,2013a).ThissuggeststhatsaRNAsmightalsoact co-transcriptionally, possibly by destabilizing secondary struc-tures peculiar tonascent pre-mRNAsand thus promotingtheir elongation.

Remarkably, saRNAs delivery also leads to extensive and complexchangesofthecovalentepigeneticpro_fileofchromatin. Dependingonthegeneofinterest,epigeneticchangestriggeredby saRNAsincludedanincreaseofH3K4me2and/orH3K4me3,H3ac and H4ac,aswell asa decreaseofH3K9me2 and/orH3K9me3, H3K27me3, H3K9ac and H3K14ac(Majid et al., 2010; Turunen etal.,2009;Matsuietal.,2013;Huangetal.,2012;Janowskietal., 2007;Modarresietal.,2012;Lietal.,2006).Conversely,nochange inCpGmethylationwasreportedatall.

Inadditiontocovalentepigeneticchanges,thefunctionallink between RNAa and chromosomal DNA looping is particularly intriguing.AsfoundbyChromosomalConformationCapture(3C), the50andthe30endsofthePRgenephysicallyinteractinMCF7 cells. While not affectingthe degreeof this intrageniclooping, saRNAs captured ncRNAs stemmingfrom the otherend in RIP assays whendirected againstthe twodifferent geneends (Yue etal.,2010).Moreremarkably,inA549cells,targetingoftheCOX2 promoter by saRNA12 induced looping of the DNA interposed between COX2 and Phospholipase A2 (PLA2G4A), whose protein productisrequiredforthesynthesisofarachidonicacid,theCOX2 substrate.With thismechanism, COX2and PLA2G4Apromoters, whichlieonthesamechromosomealmost150kbfarfromeach other in divergent orientation, got into close tridimensional vicinity, leading to coordinated transactivation of both genes (Matsuietal.,2013).

Inconclusion,awealthofinformationabout“basic”molecular eventstriggeredbysaRNAsisavailablenowadays.However,wedo notknowthegeneralcausalorder,ifany,linkingoneanother.In particular,wepresentlyignoreiftheepigeneticeventsexemplified aboveare propedeutictothetranscription arousaloccurringin RNAa,orareaconsequenceofit.Furtherindepthstudieswillbe requiredtosolvethisissue.

6.5.RNA-programmableNMHVtranscriptionfactors

To expand the repertoire of molecular tools available for therapeuticstimulationofgenetranscription,oneofourgroups recently developed a novel class of artificial transactivators, termed NMHVs (an acronym standing for Nuclear localization signal
MS2coatproteinRNAinteractingdomain
HAepitope
(3x)VP16 transactivating domain)(Fig.2)(Fimiani et al.,2015). TheseareRNA-programmableenzymeslikeCRISPR-TFs,however theydifferfromCRISPR-TFsinseveralaspects.First,theyrequire longerRNAbaitsforeffectiveandspeci_fictargetrecognition(60 basesvs20bases).Moreover,comparedtoCRISPR,NMHVs(1)are >7-foldssmaller,(2)elicitanormalizedtranscriptionalgainaround twofold,and(3)selectivelystimulatetranscriptionofactivegenes, whilenotaffectingsilentones.Thesetraitsareverydifferentfrom thecorrespondingfeaturesofmorepopular,RNA-programmable CRISPR-TFs, which are very large, can switch on silent genes, possiblybecauseoftheirintrinsichelicaseactivity(Chengetal., 2013;Kearnsetal.,2014),andeasilyachieveveryhighexpression gains, even >500-folds (Perez-Pinera et al., 2013). For these reasons,CRISPR-transactivatorswouldbehardlyemployablefor scalable and finely tuned correction of haploinsufficiencies. Conversely,broadinvivodeliveryofNMHV-TFsseemtobebetter

Fig.1.PutativemechanismsofsaRNAs.(A)saRNAsmaydriveAgo2-dependent degradation of nascent, noncoding RNAs, in charge of recruiting W and Z polypeptidictrans-repressors tothegene ofinterest (goi).(B,C)Alternatively, saRNAs may drive Ago2-dependent recruitment of X and Y polypeptidic transactivatorstothegoi.ThesaRNAmaydirectlyinteractwithlocallydenatured goi-DNA(B)orwithanascentncRNA,stemmingfromgoichromatin(C).

suitable for clean and scalable transcriptional rescue of these insufficiencies. This prediction, as well as a more thorough assessment of NMHV off-targeting effects, currently waits for experimentalvalidation.

6.6.SINEUPs

The use of long non-coding RNAs as biotechnology and therapeutic tools is still at infancy. Some of us have recently shownthatASUchl1,alncRNAantisensetothemouseortologueof thehumanUchl1/Park5gene,increasesUchL1proteinsynthesisat post-transcriptionallevel(Carrierietal.,2012).ASUchl1is a50

head-to-headdivergentantisenselncRNA,thatpartiallyoverlaps withUchl1mRNAcoveringinitiatingAUG.Whenoverexpressedin murinedopaminergiccellline,ASUchl1isabletoincreaseUchL1 protein product without affecting its mRNA levels. Under physiological conditions, AS Uchl1 is retained in the nucleus. Uponrapamycintreatment,ASUchl1shuttlesfromthenucleusto the cytoplasm with an unknown mechanism. Once in the cytoplasm,ASUchl1 induces Uchl1 mRNAassociationto heavy polysomes,thusincreasingitstranslation.Wecoulddemonstrate thatASUchl1activitydependsonthecombinationoftwo RNA elements:atthe50end,theoverlappingregionisindicatedasthe Binding Domain (BD); while the 30 non-overlapping region containsanembeddedinvertedSINEB2element,whichrepresents theEffectorDomain(ED)(Fig.3).TheEDisrequiredforprotein up-regulationfunctionofASUchl1andtheBDdictatesitsspecificity towards Uchl1 mRNA. By swapping BD sequences, a synthetic lncRNAcanbedesignedinwhichASUchl1activityisre-directedto target exogenous transcripts, as those encoding for Green Fluorescent Protein (GFP) (Carrieri et al., 2012; Zucchelliet al., 2015b).Therefore,ASUchl1canbeconsideredtherepresentative member of a new functional class of AS lncRNAs that utilize embeddedinverted SINEB2 elements to increase translation of partially overlapping protein-coding genes acting on their endogenouslyexpressed mRNAs(Carrieriet al., 2012; Zucchelli etal.,2015b).Thesenaturalandsyntheticmoleculeswerenamed SINEUPssincetheirfunctionrequirestheactivityofanembedded inverted SINEB2 sequence to UP-regulate translation (Zucchelli etal.,2015a,2015b).SINEUPsarethusthefirstexampleof gene-specificinducersofproteinsynthesis.SINEUPmodularstructure can be employed to artificially engineer their BD and design syntheticSINEUPstospecificallyenhancetranslationofvirtually anytarget geneofinterest. SyntheticSINEUPssofarhave been proveneffectivewithanumberoftargets,includingGFP(Carrieri et al., 2012), FLAG-tagged proteins (Zucchelli et al., 2015b), secretedrecombinant antibodiesand cytokines(Patrucco etal., 2015),thusshowingSINEUPtechnologyisscalable,onceprovided the required target recognition with BDs. Most importantly,

syntheticSINEUPscanactonendogenousmRNAsinvitroandin vivo,asdemonstratedbyspecificSINEUPsdesignedtotargetgenes associated toneurodegeneration(PARK7/DJ-1 for PD)(Zucchelli etal.,2015b)andbraindevelopmentaldisorders(Indrierietal., 2016).

Overcompetingtechnologiesaimedtoincreasequantitiesofa proteinof interest,SINEUPspresentthree majoradvantages:1) Inducea2-to-5-foldup-regulationthuslimitingsideeffectsdueto exaggeratedoverexpressioningenetherapyapproach;2)Acton endogenousmRNAsinsitu,restrictingtranslationenhancementto the time and spaceof endogenous gene expression, 3) Do not triggerhereditablegenomeediting.Similarlytoothernucleic acid-basedtherapiesforbraindisorders,thefinaloutcomeof SINEUPs-basedtherapywilldependonreachingtheirtargetmRNAsinthe brain. The currentrenaissance in nucleic acid-baseddrugs will providethecorrectmoleculartoolsfortheirdelivery.

7.TheYangsideofnucleicacid-basedtherapy:applicationsto neurodegenerativediseases

7.1.Angelmansyndrome

UBE3AisanimprintedgeneencodinganE3ubiquitinligase. MaternaldeficiencyofUBE3AistheprimarycauseofAngelman syndrome,adisordercharacterizedbysevereintellectualdisability and developmental delay, withbehavioural deficits and severe speech impairment, associated to seizuresand ataxia. Patients withAngelmansyndromecarryoneintactcopyofpaternalUBE3A gene,whichisinactivatedbyanantisensenuclear-retainedlncRNA (UBE3A-AS).ASOtreatmentwasdevelopedtotargetUBE3A-AS(as carried outinAntagoNAT-basedtherapy)tocorrect forsilenced UBE3A expression in Angelman Syndrom mouse model (Meng etal.,2015).Intra-cerebroventricularinjectionofsingle-doseASO was well tolerated. After ASO treatment (4 weeks), UBE3A-AS expressionwasreducedby60–70%andABE3AmRNAincreased2– 5 fold in brain and spinal cord. Correction of phenotypes was evidentbutnotcomplete.

7.2.Spinalmuscularatrophy

Spinal muscular atrophy (SMA) is an autosomal recessive disorder,causedbymutationsintheSMN1genethatresultina deficiency ofSMN proteinthat ultimatelycauseneuromuscular defects.TheseverityofSMAisdictatedinpartbythecopynumber oftherelatedduplicatedgeneSMN2,whichhasacriticalmutation in theexonic splicing site of exon 7.Therapeutic strategies are aimedat replacing insufficient gene dosagebyAAV delivery of SMN1cDNA.Analternativestrategy istouseASOstore-direct splicingofparalogueSMN2RNAtoboostexpressionoffunctional SMNprotein.Intra-cerebralventricleinjectionintoamousemodel ofsevereSMAresultedinincreasedSMNproteinlevels,increased

Fig.2. SchematicsofRNA-programmableNMHVtransactivators.NMHV transacti-vatorharborsapolypeptidicNMHVapofactorandanRNAcofactor.Theformer includesanNLS2(nuclearlocalizationsignal,2x),anMS2-likeRNAbindingdomain

(RBD),anHAhemaggulitininepitopeandaVP163TransActivatingDomain(TAD).

ThelatterresultsfromamultimerizedMS2-interactingfinger(MFn)anda60–180

baselongbait,fortherecognitionoftranscriptionstartsitesequences.

Fig.3. SchematicrepresentationoftargetmRNAandtarget-specificSINEUP.50_to30

orientation of sense mRNA and antisense SINEUP is indicated. Black arrows representtranscriptioninitiationandorientation.Structuralelementsoftarget protein-codingmRNA(green):50untranslatedregion(50UTR),codingsequence (CDS)and30untranslatedregion(30UTR).InitiatingAUGisshowninred.Structural elements of target-specific SINEUP (purple): Binding Domain (BD), SINEUP sequence thatoverlaps,in antisense orientation,to the sense protein-coding mRNA; Effector Domain (ED), inverted SINEB2 element (invB2) thatconfers activationofproteinsynthesis.(Forinterpretationofthereferencestocolourinthis figurelegend,thereaderisreferredtothewebversionofthisarticle.)