medRxivpreprintdoi:

/10.1101/2023.02.21.23285822

;thisversionpostedFebruary24,2023.Thecopyrightholderforthispreprint

(whichwasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaythepreprintinperpetuity.

Itismadeavailableundera

CC-BY4.0Internationallicense

.

InVivoCRISPRGeneEditinginPatientswithHerpesStromalKeratitis

Authors:AnjiWei1?,DiYin2?,ZimengZhai1?,SikaiLing3,HuangyingLe2,LijiaTian1,

JianjiangXu1,SorenRPaludan4,YujiaCai2*,JiaxuHong1,5,6*.

Affiliations:

1DepartmentofOphthalmologyandVisualScience,Eye,andENTHospital,Shanghai

MedicalCollege,FudanUniversity;Shanghai,China.

2KeyLaboratoryofSystemsBiomedicine(MinistryofEducation),ShanghaiCenterfor

SystemsBiomedicine,ShanghaiJiaoTongUniversity;Shanghai,China.

3BDgeneTherapeutics;Shanghai,China.

4DepartmentofBiomedicine,AarhusUniversity;Aarhus,Denmark.

5ShanghaiKeyLaboratoryofVisualImpairmentandRestoration,ScienceandTechnologyCommissionofShanghaiMunicipality;Shanghai,China.

6ShanghaiEngineeringResearchCenterofSyntheticImmunology;Shanghai,China.

?Theseauthorscontributedequallytothiswork.

*Correspondingauthor.Email:JH:Jiaxu.hong@,YC:yujia.cai@.

Abstract:InvivoCRISPRgenetherapyholdslargeclinicalpotential,butthesafetyandefficacyremainlargelyunknown.Here,weinjectedasingledoseofHSV-1-targetingCRISPR

formulationinthecorneaofthreepatientswithsevererefractoryherpesstromalkeratitis(HSK)duringcornealtransplantation.Ourstudyisaninvestigatedinitiated,open-label,single-arm,

non-randomizedinterventionaltrialatasinglecenter(NCT04560790).Wefoundneither

detectableCRISPR-inducedoff-targetcleavagesbyGUIDE-seqnorsystemicadverseeventsfor

18monthsonaverageinallthreepatients.TheHSV-1remainedundetectableduringthestudy.

OurpreliminaryclinicalresultssuggestthatinvivogeneeditingtargetingtheHSV-1genomeholdsacceptablesafetyasapotentialtherapyforHSK.

One-SentenceSummary:OurstudyisthefirstinvivoCRISPRtherapyfortreatinginfectiousdiseaseandthefirstvirus-likeparticle(VLP)-deliveredgenetherapy,reportingclinicalfollow-upto21monthsinHSKpatientswithoutseeingvirusrelapse,HSKrecurrence,andCRISPR-associatedsideeffects.

NOTE:Thispreprintreportsnewresearchthathasnotbeencertifiedbypeerreviewandshouldnotbeusedtoguideclinicalpractice.

medRxivpreprintdoi:

/10.1101/2023.02.21.23285822

;thisversionpostedFebruary24,2023.Thecopyrightholderforthispreprint

(whichwasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedmedRxivalicensetodisplaythepreprintinperpetuity.

Itismadeavailableundera

CC-BY4.0Internationallicense

.

Introduction:

Herpessimplexvirustype1(HSV-1)isacommonhumanvirus,withaglobalseroprevalenceof50–90%(1-3).OcularHSV-1infectionisthemajorcauseofherpeticstromalkeratitis(HSK)

whichisoneoftheleadingcausesofinfectiousblindnessindevelopedcountries(4).

Approximately,40,000peopledevelopvisualdisabilityamongthe1.5millionnewcasesof

ocularHSVinfectioneachyear(4).Afterprimaryinfectioninthecornea,HSV-1establishesalatentreservoirinthetrigeminalganglia(TG),whichcanbereactivated,leadingtothe

recurrence(5).

Currently,novaccineisavailableagainstHSVinfection(6).Acyclovir(ACV)andanalogsthattargettheviralDNApolymerasearethefirst-choicetreatmentsforHSK(7).However,asthe

antiviralmoleculesdonotchangetheexistingviralDNA,recurrencesarestillcommon.

Additionally,resistancetoACVhasbeenassociatedwithlongerdiseasedurationandsubsequentfailureofprophylaxisinpatientswithrecurrentocularHSVepisodes(7,8).Otherstrategies,

includingantibodies,peptides,andsmallmoleculesarestillunderdevelopment(9).Corneal

transplantationisrecommendedforHSK-inducedcornealleucomaorperforation.However,thepostoperativeprognosisiscompromisedbyhighratesofvirusrecurrence(10).Accordingto

previousstudies,thereisahighincidenceofherpetickeratitisrecurrencefollowingpenetratingkeratoplasty(PK)-evenwiththetopicalacyclovir,therecurrentrateisstillover55%(10,11).VariousstudieshavereportedtherecurrencerateofHSVinfectioningraftswithprophylacticoralacyclovirtherapyrangingfrom12%to33%(11-13).Notably,systemicACVhasbeen

associatedwithkidneyinjuryandneurotoxicity(11,14).Essentially,neitherthedrugsnor

surgicaltreatmentscandiminishthevirusinthecorneasorTGreservoirandcleavethevirusgenomedirectly,whichdrivestheexplorationofnext-genantiviralstrategieswith

meganucleasesandCRISPR(15-17).

CRISPRhasbeenremarkablysuccessfulinpreclinicalresearch(18-22).Sofar,ithasbeen

appliedinclinicaltrialsfortreatinghemoglobindiseases,cancers,andHIVinfection(23-27).

However,thepublishedtrialsareallexvivoexcepttherecentreportbyGillmoreetal.inwhichCRISPRwasusedforinvivotreatmentoftransthyretinamyloidosis(28).Still,thebroaderin

vivosafetyandefficacyprofileofCRISPRremainstobeestablished.ToovercomethedeliveryobstacleforinvivoCRISPRtherapy,severalgroupsincludingourshavereportedengineered

virus-likeparticleswhichareabletotransportmRNAorribonucleoprotein,allowingtransient

geneediting(29-31).OurgrouphasdevelopedanmRNA-carryinglentiviralparticle(mLP),

capableofincorporatingmRNAofinterestsuchasSpCas9intheproducercellsviathe

interactionbetweenaptamerinthemRNAandadaptorintheviralstructuralprotein(32).We

furtherdesignedtheSpCas9mRNA-carryinglentiviralparticle,termedHSV-1-erasinglentiviralparticle(HELP),tospecificallycleavetwoessentialgenesfortheHSV-1lifecycle,UL8and

UL29,forHSKtherapy(33).AsingleintracornealinjectionofHELPsignificantlydiminishedHSV-1inthecorneasandTGinmice(33).

AlthoughwehaveshownHELPefficientlycontrolledtheHSVreplicationinvitro,inanimal

modelsandexvivohumancorneas,itmaybedifficulttoclearallthevirusesinthereservoirinhumans.Wehypothesizedthatreducingthevirusloadtoacertainthresholdwillchangethe

balancebetweenvirusandhostsothattheimmunesystemcancontroltheremainingvirusandachieveafunctionalcure(34,35).Here,wereporteddatafromaclinicaltrialevaluatingthe

safetyandefficacyofonedoseHELPinjectionduringthepenetratingkeratoplastyforthreepatientswithsevererefractoryHSKandacutecornealperforation(Figure1),allthreehad

completedthe12monthsfollow-up(21months,18monthsand14monthsforpatient1,2and3,respectively).

Results:

PreclinicalResults

ThepresenceofHSV-1inthecorneasoftheparticipantwasdeterminedbyquantitative

polymerasechainreaction(qPCR)analysisoftheviralgenomeintear-swabbedsamples.The

positiveresultwasapreconditionforenrollment(Table1).HELPwasgivenbyintrastromal

injectionatadoseof2.4μgp24duringpenetratingkeratoplastyillustratedinFig.1.The

perforatedcorneawasremovedfromthepatientduringtheoperationforsubsequentfluorescencemicroscopyandimmunohistochemistryanalysis.TheviralcapsidproteinVP5wasdetectedin

thecorneasofthreepatientsbyconfocalimaging(ExtendedFigure1).AsHSKisthe

consequenceofexcessivevirus-inducedcornealinfiltrationofinflammatorycells(36),we

thereforestainedtheremovedcornealbuttonforTcells(CD4+andCD8+),myeloid-derived

cells(CD11b+)andmacrophages(F4/80+).Immunohistochemistryshowedtheirinfiltrationinthecorneastromaofthreepatients(ExtendedFigure2).WesequencedHSV-1strainsisolatedfromthreeparticipantsforgenesencodingthymidinekinase(TK)andDNApolymerase(DNApol).WefoundchangesinaminoacidsinTKorinDNApol,buttheywerenotdrug-resistancemutations(FigureS1fromSupplementalInformation).Additionally,weusedaHSV-1infectedhealthycorneatoevaluatethefunctionalityofCRISPRandfoundasignificantreductionof

HSV-1genomeandviablevirusesinadditiontothebaredetectableVP5antigen(Figure2A-E).Also,wefounddirectevidenceofviralgenomecleavageinUL8andUL29lociwithindel

frequencies28.8%and14.4%,respectively,bydeepsequencing(Figure2F).

PatientDemographicsandOutcomes

Patient1

Patient1wasaseniormaleinhisearly70swhowasadmittedinNovember2020forcorneal

perforationduetorecurrentHSKintherighteyeandhadinitialHSKnearly30yearsagowitharecurrenceintervalofevery1-2years.Hehadbeenprescribedahighdoseofantiviral

medicationsyetstillfailedtoamelioratethekeratitis.Hislefteyewasdiagnosedwithtraumaticcornealleucoma.Onexamination,uncorrectedvisualacuitywaslightperceptionforbotheyes.Theslit-lampimageoftherighteyeshowedanill-definedcentralcornealulcerofabout3mm×3mmwithirisincarceratedintheperforation.Thelefteyeshowedacloudycorneawithout

conjunctivalcongestion.Patient1hadmildanemiaandahistoryofdiabeteswithaslightlyhighglycatedhemoglobinof6.2%.

Topicalganciclovireyegelwasdiscontinuedforthepatientonedaybeforetheuncomplicated

PK.DuringthePKoperation,theiriswascarefullyseparatedandseverelensopacitywasfoundinthesurgicaleye.Patient1receivedHELPinjectionaccordingtotheprotocol(Figure3A).

Topical0.5%levofloxacinand1%prednisoloneacetatewereadministeredthreetimesperdayafterthesurgery.Postoperativehyphemawasidentifiedimmediatelyafterthesurgery,which

wasabsorbedonday7.Hisvisualacuityoftherighteyehasremainedfingercountowingtotheseverecataractsinceday3.At6monthsafterPK,patient1developedanuncontrolledcorneal

ulcerrelatedtoneurotrophickeratopathy(NK),whichmainlyresultedfromthelong-lasting

damagetothecornealsensorynerveinducedbychronicHSVinfection.Inaccordancewithhisdecreaseincornealsensitivity,invivoconfocalmicroscopy(IVCM)showedsignificantly

impairedcornealsubbasalnervesinthecentralcorneagraftatthelatestfollow-up(Extended

Figure3).Tofurtherimprovethepatient’svision,wethereforeconductedthesecondcorneal

transplantationandphacoemulsificationwithintraocularlensimplantationonhisrighteye.Hehadregainedanuncorrectedvisualacuity(decimal)of20/100inthesurgicaleyebythetimeofdischarge(Figure3B).Thepostoperativeintraocularpressurewaswithinthenormalrange

(TableS1fromSupplementalInformation).Opticalcoherencetomography(OCT)showedarelativelyshallowanteriorchamberinpatient1(ExtendedFigure4).HiscornealgrafthadremainedtransparentsincethesecondPKafter12monthspost-injection(Figure3A).

Beforetreatment,boththecornealbuttonandaqueoushumourwerediagnosedpositiveforHSV-

1usingaTriplexHSV-1DNADiagnosticKitwithcyclethreshold(Ct)valuesof21.35and

28.37,respectively(TableS1).However,tearswabshavemaintainednegativesincehis2-monthvisit(Table1).Interestingly,thesecondcornealtransplantationinthesixthmonthgaveusa

uniqueopportunitytoexplorewhethertheHELPcandiminishtheHSV-1genomeinvivoby

examiningtheexcisedcornealbutton.Indeed,wefoundVP5signalsneitheraroundtherimnorinthecenteroftheremovedcornealbuttonviafluorescencemicroscopyand

immunohistochemistryanalysis(ExtendedFigure5and6).

Patient2

Patient2wasamaleinhisearly50swhohadanacutecornealperforationintherighteyecausedbyrecurrentHSK.Hisrighteyehadbeensufferingfromrepeatedrednessandpainformorethanadecadewitharecurrenceintervalof2-3months,whichcouldnotbeeffectivelycontrolledby

variousantiviralandglucocorticoideyedrops.FivedaysbeforeadmissioninJanuary2021,thepatientcomplainedofsharp,suddenpainandfluidleakageintherighteye.Onexamination,

uncorrectedvisualacuity(decimal)was20/20inthelefteyeyetonlyhandmotionintheright

eye.Theslit-lampimageoftherighteyerevealeda4mm-diameterlowerparacentralcorneal

ulcerwithapinpointleakagebutnohypopyonintheanteriorchamber.Thelensoftherighteyewasslightlycloudy.Hislefteyewasunremarkable.Patient2hadahistoryofchronicB-relatedhepatitisandhyperlipidemia.

UncomplicatedPKwasperformedforpatient2whoalsoreceivedHELPinjectionsubsequentlyaccordingtotheprotocol.Afterthesurgery,topical0.5%levofloxacinand1%prednisolone

acetatewereappliedthreetimesadaywhereasthetraditionalacyclovireyedrops(everytwo

hours)andoraltablets(twicedaily)werediscontinued.Onedayaftersurgery,topical

prednisoloneacetatewasincreasedtofourtimesdailyduetocornealgraftedema.Twodays

later,intraocularpressureoftherighteyewas11mmHgandvisualacuityraisedtofingercount.OCTatthistimeconfirmedaflatirisandopenanteriorangleinalldirections(ExtendedFigure7).Atlatervisits,hisvisualacuityimprovedto20/167immediatelyatonemonthvisitand

stabilizedas20/67at3monthsafterthesurgery(Figure3B).InthesixthmonthafterPK,patient2wasdiagnosedofStaphylococcalendophthalmitiswhichwasconfirmedbybacterialand

fungalcultures(TableS2)andsubsequentlycontainedbythecombinationofvitrectomyand

antibioticinjection.Hisuncorrectedvisualacuityhaddecreasedto20/133becauseofthe

secondarycataractinthesurgicaleye(Figure3B).IVCMrevealedpartialregenerationofcornealnervesathis12month-follow-ups(ExtendedFigure8).Norelapseofherpetickeratitisorgraft

rejectionwasidentifiedatthefinalvisit.

Beforetreatment,thepatientwaspositiveforHSV-1byqPCRanalysisoftheremovedcornealtissue(Ct=26.34)andtearswabs(Ct=35.01),andtheaqueoushumourwhichwasweak-positive(Ct=36.50)(TableS1).Inthesubsequent12-and18-monthfollow-ups,however,wefoundthetearswabssamplesduringthepostoperativevisitsshowedthattheHSV-1testswereallnegativebyqPCRexamination(Table1).

Patient3

Patient3wasamaleinhislate60swhohadsix-yearofHSKandeventualcornealperforationinthelefteye10daysbeforeadmissioninMay2021.HisHSKrelapsedevery2-3months.The

sameeyehadphacoemulsificationwithintraocularlensimplantation8yearsago.Hisrighteyewasunremarkable.Onexamination,uncorrectedvisualacuity(decimal)was20/50intherighteyeandhandmotioninthelefteye.Conjunctivacongestionandlimbalneovascularizationweresevereinhislefteye,withagrayulcerof5mm×5mmatthecenteroftheopaquecornea.

Subacutecorneaperforationandhypopyonindicatedahigh-riskcornealtransplantation.OCTconfirmedpartialperforationonthecornea(ExtendedFigure9).Patient3hadahistoryof

hypertension.

AftertheuneventfulPKandHELPinjection,thepatienthadbeenprescribed0.3%tobramycin

and0.1%dexamethasoneophthalmicointmentfourtimesdaily.Brinzolamidetimololdrops

(twiceperday)wereprescribedforhiselevatedintraocularpressureowingtopostoperative

hyphema.Ganciclovireyegel(fourtimesdaily)andacycloviroraltablets(twicedaily)were

discontinuedaftertreatment.Onedayafterthesurgery,0.5%cyclosporineeyedropswereaddedtosuppressinflammationoftheocularsurface.Duetotheintraocularlensopacityinducedby

cornealperforation,thevisualacuityofpatient3hadbeenhandmotionsincePKoperationandfingercountat12-month(Figure3B).Onhisthirdmonthfollow-up,weobservedcornealedemaandinflammatorycellinfiltrationaroundthecornealsutures,indicatinganoccurringgraft

rejectionwhichwascontrolledafterreceivingtopicaltreatmentforthegraftrejectionand

secondaryglaucoma.PostoperativeB-scanultrasonographysuggestednosignificantabnormalchangesintheretina(FigureS2).

ThoughwedidnotdetectHSV-1inaqueoushumourinpatient3beforeCRISPRtreatment,thepatientwasdiagnosedasHSV-1positivefordetectingHSV-1inthetearswab(Ct=29.5)andtheremovedcornealtissue(Ct=34.9)duringPKbyqPCR,andtheviralVP5antigenusingconfocal

imaging(Table1,TableS1andExtendedFigure1).However,inthe12-and14-monthfollow-ups,wefoundtheswabsamplesduringthepostoperativevisitswereallnegativeforHSV-1

(Table1).

AdditionalSafetyAssessment

ThepotentialHELP(CRISPR)-relevantsideeffectsaretheimmuneresponseattackingthe

CRISPR-transducedcellsexpressingCas9andoff-targetcleavages.ELISAshowedintrastromalinjectionofHELPdidnotprovokeananti-vectorimmuneresponseasnop24-specificIgGwasinduced(ExtendedTable1).Interestingly,byexaminingthebloodsamples,allthepatientswereSpCas9-positivebeforetreatmentwhiletheCas9-specificIgGwasnotsignificantlyenhanced

aftertreatment(FigureS16).Toanalyzethepotentialoff-targeteffectsofourvirus-targeting

HELPinthehumangenome,weperformedGUIDE-seqtorevealgenome-wideintegrationsofdouble-strandedoligodeoxynucleotidesinthedouble-strandDNAbreakscausedbyCRISPR

(Figure3C)(40).However,insubsequentdeepsequencing,wedidnotdetectindelsonthethreeGUIDE-seqidentifiedsitesinthedetachedepithelialcellsfromwipingthecorneaofthepatients1weekafterthetreatmentaswellasin293Tcells(Figure3D,E).Fortherest,weperformeda

completeophthalmicexaminationonthreepatientsafterHELPtreatment(Supplementary

Information).Theocularadverseevents(AE)weobservedincludedcornealgraftedema,

hyphema,post-herpesNK,concurrentcataractandsecondaryglaucoma,whichweremainly

attributedtothehigh-riskpenetratingkeratoplastywithacutecornealperforation(37-39)(TableS2).B-scanultrasonographyofthepatientson7days,1month,6monthsand12monthspost-

injectionshowednoabnormalchangesinthevitreousbodyandretina(FigureS2-S4).Retinal

OCTdemonstratedrelativelyintactfundusinHELP-injectedeyes(FigureS5,S6).Additionally,wefoundnoapparentchangeinrodorconeresponsesintheparticipantsbythe

electroretinography(ERG)(FigureS7-S9).NosystemicAEswerefoundduringthestudyperiod.

Discussion:

WereportthreerefractoryHSKcaseswithacutecornealperforationreceivinginvivoCRISPRtherapeutics.AfterasingleintrastromalinjectionofHELPincombinationwithcorneal

transplantation,cornealgraftsandtearswabsinthe3patientswerestillfreeofviralrelapseat

theirlastvisitswiththeaveragefollow-upsreaching18monthseventhoughwediscontinuedtheantiviraltherapyaftertheCRISPRinjection.IntheHSV-1infectedhealthycorneas,HELP

efficientlydiminishtheHSV-1intermsofgenome,viablevirusesandantigen(Figure2).

Moreover,wedidnotdetectHSV-1intheremovedcornealbuttonfrompatient1at6months

postHELP-treatmentduetoneurotrophickeratopathy,addingsupportingpiecethattheHELP

coulddiminishtheHSV-1inthecorneas(ExtendedFigure5).ThelaboratorydatasuggestednoCRISPRoff-targetcleavageinthehumangenomeorCas9andvector-specificimmuneresponseinducedbyintrastromalinjectionofHELP.TheseresultssuggestthatHELPmightbean

effectivestrategyinrestrictingHSV-1replicationinthehumancorneaswithnoremarkableCRISPR-relatedsideeffects.

AlthoughtheHSV-1DNAlevelsbecameundetectableimmediatelyaftertreatmentinpatient2

and3,theviruswasstilldetectedinthefirstmonthforpatient1,butnotatlatertimepoints.Wereasonseveralmechanismsmayberesponsibleforthisphenomenon.ItislikelythatHELP

reducesviralloadbytargetingbothproductivereplicationandthelatentreservoirwithout

achievingfullelimination,especiallyincaseslikepatient1whohadamuchhighervirusload

thanpatient2andpatient3(TableS1andExtendedFigure1).Therefore,theacutestress-

associatedhostresponseislikelytoaugmentviralreplication(41,42).Second,immune

activitiesarealsolikelyrequiredtofullyinhibitthevirusaftertheHELPtreatment.Patient1wasinhisseventiesandthereforemighthavearelativelyweakimmunesystemnotfullycapableofcontrollingresidualvirusesinthecorneasandTGthefirstmonthsaftertreatment.Tobalancethebenefit-riskofthefirstantiviralCRISPRtherapy,patient1waschosenbecausehehadveryhighpreoperationalviralloadsinthecornea,aqueoushumourandtearswabs.Therefore,itisnot

surprisingthatHELPwasunabletocleartheviruscompletelyinthispatient,butonlyreducethevirusburdeninvivo.However,asthecorneaofpatient1wastransparentattheone-monthvisitwithoutsignsofrecurrence,wecontinuedantiviraldrugs-freeprotocolonthepatient.Notably,

hisswabsofbothcorneaandtearfilmremainednegativeforHSV-1from2-monthtothefinal

visit.Accordingtothecurrentstudy,patient2and3,whohadlowerpre-operationalHSV-1loadcomparedwithpatient1,hadobtainedamorethoroughHSV-1clearancebyHELPjudgingfrom

theirvirustests.FuturestudiesenrollingmorepatientswithdifferentdegreesofHSKwill

answerwhetherpatientswithlowerpre-operationalcornealvirusloadthanthethreepatientsinthisstudywillhaveanevenmorebeneficialoutcomeafterHELPtreatment.

TheparticipantsenrolledinourstudywereallexperiencingrecurrentHSKepisodesfordecades,raisingthepossibilityofdrug-resistantHSV-1infection.Accordingly,wefoundrespectiveratherthansimultaneouslychangesofaminoacidsinTKandDNApolinthepatients’isolatesthat

weredifferentfromboththeKOSand17syn+strainsofHSV-1(FigureS1),suggestingthattheconstantreleaseofHSV-1fromthereservoir,insteadofdrugresistance,mayhavecontributedtotherefractoryHSKinthesepatients.Todate,theHSV-1reservoirinTGshasstillbeen

untargetablewithantiviraldrugs.WehavepreviouslyshowninthepreclinicalmodelthatHELPiscapableofmodulatingthevirusinthereservoirviaretrogradetransportationofCRISPRtoTG(33).High-riskcornealtransplantationfortheperforatedcorneaispronetoHSKrecurrenceandcornealrejection.Therefore,thediscontinuationofantiviraltherapyafterPKcouldmake

participantsmorevulnerabletoHSV-1recurrencethanotherpatients.However,amongthe21

testsofHSV-1onthreeparticipants,except2testsinthefirstmonthofpatient1werepositivewithoutanyclinicalsymptoms,alltheresttestswiththelongestfollow-upupto21monthsaftertreatmentwerenegative.Thus,allthreeparticipantshadnoHSKrecurrenceduringthisstudy

althoughthespecificroleofHELPonHSV-1inthehumanTGreservoirremainselusive.

Wesummarizedinatableforalltheadverseeventsdividedbythepatientsandbyfollow-up

visits(supplementaryinformationTableS2).AlthoughHELPtreatmentmaynotbefully

excludedfromtheobservedcomplicationsduetothelimitedcasesandlackofcontrols,thiswasveryunlikelyexceptforcornealedema.Numerouscomplications,suchasgraftrejection,

hyphema,glaucoma,orcataractcouldbefoundinpostoperativetreatmentofpenetrating

keratoplastydespitetheprogressofsurgicaltechniques,especiallyinthehigh-riskcaseswith

acutecornealperforation(37).HyphemainPatient1ismainlyattributedtothetearoftheiris

duringthesurgery,whichfinallyresolved.Cataractinourthreecasesmayhappenbecauseof

acutecornealperforationandtheusageofcorticosteroidafterthesurgery.Theincidenceof

glaucomainPatient1and3isnotrareaftercornealtransplantation.Severaletiologicfactors

havebeenidentified,themostcommonbeingsynechialangleclosureandcorticosteroid-inducedIOPelevation.IVCMrevealedHSV-relatedcornealepitheliumabnormalityandlownerve

densityinallthreeparticipants,indicatingtheirhighsusceptibilitytopost-herpesNK.DamagetothesensorynerveendingofthetrigeminalnervebyHSV-1couldresultindecreasedcorneal

sensitivity,lossofepithelialintegrityandcornealulcerationinseverecases(43).Thispossibly

couldexplainwhypatient1,aseniormalewith30yearsofHSKhistoryultimatelydeveloped

NK6monthsafterPK.TheendophthalmitisofPatient2stemmedfrombacterialinfection,

identifiedasGram-positivecocci(Staphylococcuscaprea)usingbacterialculture.TheHELP

productwasproducedandstoredunderGMP-likeregulation,withaqualitycontrolreport(TableS14).Additionally,theHELPwasinjectedintothecorneaofPatient2whichremained

transparentduringthe18-monthfollow-up.Tosumup,thepotentialHELP-relevantclinicalAEswereminimalafterCRISPRtreatment.

ImmuneresponsesagainsttheviralvectororCas9canbeproblematicforinvivoCRISPRgenetherapy(44,45).Inourstudy,novector-specificIgGwasdetectedaftertheHELPtreatment.

Indeed,prednisoloneacetatehasbeenprescribedthroughouttheentirefollow-upcourseofthe

patientsasinstructedbythestandardsforcornealtransplantationtopreventrejection,whichmayhaveplayedaroleintherestrainedinflammatoryandimmunereactionsinthepatients(9).

Additionally,wehavepreviouslycharacterizedthatHELPislowimmunogenic(33),therefore,boththelowimmunogenicityandprednisoloneacetatemayhavecontributedtotheabsenceofinflammatoryandimmunereactionsaftertheHELPtreatment.Finally,thelackofHELP-relatedadverseeffectsmayalsobeduetothetransientnatureofSpCas9mRNAdeliveredbyHELP.

HELPwasoriginallydesignedforasingledosageadministration.Inthecaseofrepeateddosing,theeffectivenessofHELPmaybecompromisedifthevector-specificIgGantibodieswere

induced,astheymayblockHELPattheentrylevel.However,theintracornealinjectionmaynotbeaneffectiveroutetoinduceanti-vectorIgGasallthreepatientswerep24negativeafterHELPtreatment(ExtendedTable1).Additionally,repeatedadministrationmaynotcausesafety

problemsforHELPastheSpCas9expressedfrommRNAexistsonlyforseveraldays,thusminimizingthechancetobeattackedbycytotoxicTcells(32).

Inconclusion,ourclinicalresultsfromthreeHSKpatientswithanaverageof18-monthfollow-upafterreceivingHELPsuggestthatinvivoCRISPRgeneeditingtargetingtheHSV-1genomeholdspromiseasasafeantiviraladjuvanttherapyforwhichtheefficacymayfurtherbe

improvedincombinationwithexistingantiviralmoleculessuc