EEEffffffeeeccctttsssooofffcccaaatttiiiooonnniiicccllliiipppooosssooommmeeesssooonnniiinnnvvviiitttrrroooaaannndddiiinnnvvviiivvvoootttrrraaannnsssddduuuccctttiiiooonnnmmmeeedddiiiaaattteeeddd

bbbyyyaaadddeeennnooovvviiirrraaalllvvveeeccctttooorrrsss

YYYeeeooonnn---KKKyyyuuunnngggLLLeeeeee

TTThhheeeGGGrrraaaddduuuaaattteeeSSSccchhhoooooolllYYYooonnnssseeeiiiUUUnnniiivvveeerrrsssiiitttyyy

DDDeeepppaaarrrtttmmmeeennntttooofffBBBiiiooommmeeedddiiicccaaalllLLLaaabbbooorrraaatttooorrryyySSSccciiieeennnccceee

EEEffffffeeeccctttsssooofffcccaaatttiiiooonnniiicccllliiipppooosssooommmeeesssooonnniiinnnvvviiitttrrroooaaannndddiiinnnvvviiivvvoootttrrraaannnsssddduuuccctttiiiooonnnmmmeeedddiiiaaattteeeddd

bbbyyyaaadddeeennnooovvviiirrraaalllvvveeeccctttooorrrsss

AAA MMMaaasssttteeerrr’’’sssTTThhheeesssiiissssssuuubbbmmmiiitttttteeedddtttooottthhheeeDDDeeepppaaarrrtttmmmeeennntttooofffBBBiiiooommmeeedddiiicccaaalllLLLaaabbbooorrraaatttooorrryyySSSccciiieeennnccceeeaaannndddttthhheeeGGGrrraaaddduuuaaattteeeSSSccchhhoooooolll

ooofffYYYooonnnssseeeiiiUUUnnniiivvveeerrrsssiiitttyyyiiinnnfffuuulllfffiiillllllmmmeeennntttooofffttthhheeerrreeeqqquuuiiirrreeemmmeeennntttsssfffooorrrttthhheeedddeeegggrrreeeeeeooofff

MMMaaasssttteeerrrooofffSSSccciiieeennnccceeeiiinnnMMMeeedddiiicccaaalllTTTeeeccchhhnnnooolllooogggyyy

YYYeeeooonnn---KKKyyyuuunnngggLLLeeeeee

JJJuuunnneee,,,222000000777

TTThhhiiisssccceeerrrtttiiifffiiieeesssttthhhaaatttttthhheeemmmaaasssttteeerrr’’’sssttthhheeesssiiisssooofffYYYeeeooonnn---kkkyyyuuunnngggLLLeeeeeeiiisssaaapppppprrrooovvveeeddd...

TTThhheeesssiiissssssuuupppeeerrrvvviiisssooorrr:::YYYooonnngggSSSeeerrrkkkPPPaaarrrkkk

JJJooonnngggBBBaaaeeeKKKiiimmm:::TTThhheeesssiiisssCCCooommmmmmiiitttttteeeeeeMMMeeemmmbbbeeerrr

HHHyyyeeeyyyooouuunnngggLLLeeeeee:::TTThhheeesssiiisssCCCooommmmmmiiitttttteeeeeeMMMeeemmmbbbeeerrr

TTThhheeeGGGrrraaaddduuuaaattteeeSSSccchhhoooooolllYYYooonnnssseeeiiiUUUnnniiivvveeerrrsssiiitttyyyJJJuuunnneee,,,222000000777

- IV -

CCCOOONNNTTTEEENNNTTTSSSLLLIIISSSTTT OOOFFFFFFIIIGGGUUURRREEESSS····························································ⅥLLLIIISSSTTT OOOFFFTTTAAABBBLLLEEESSS ·····························································ⅧAAABBBBBBRRREEEVVVIIIAAATTTIIIOOONNNSSS······························································ⅨAAABBBSSSTTTRRRAAACCCTTT ·········································································ⅩⅠⅠⅠ...IIINNNTTTRRROOODDDUUUCCCTTTIIIOOONNN ···························································1ⅡⅡⅡ...MMMAAATTTEEERRRIIIAAALLLSSSAAANNNDDD MMMEEETTTHHHOOODDDSSS···································4

111...MMMaaattteeerrriiiaaalllsss ········································································4222...CCCeeellllllllliiinnneeesssaaannndddccceeellllllcccuuullltttuuurrreee···················································5333...PPPrrreeepppaaarrraaatttiiiooonnnooofffcccaaatttiiiooonnniiicccllliiipppooosssooommmeeesss··········································5444...RRRTTT---PPPCCCRRRaaannnaaalllyyysssiiisssfffooorrrCCCAAARRReeexxxppprrreeessssssiiiooonnnooonnnttthhheeesssuuurrrfffaaaccceeeooofffcccaaannnccceeerrrccceeellllllsss·················································································7

555...IIInnnvvviiitttrrroooaaadddeeennnooovvviiirrraaallltttrrraaannnsssddduuuccctttiiiooonnn···········································9666...AAAnnnaaalllyyysssiiisssooofffiiinnnvvviiitttrrroooGGGFFFPPPeeexxxppprrreeessssssiiiooonnn····································11777...CCCyyytttoootttoooxxxiiiccciiitttyyyaaassssssaaayyyooofffcccaaatttiiiooonnniiicccllliiipppooosssooommmeeesss·······························11888...IIInnnvvviiivvvooogggeeennneeetttrrraaannnsssddduuuccctttiiiooonnnaaannndddaaannnaaalllyyysssiiisssoooffftttrrraaannnsssgggeeennneeeeeexxxppprrreeessssssiiiooonnn·····················································································12

ⅢⅢⅢ...RRREEESSSUUULLLTTTSSS111...CCCAAARRReeexxxppprrreeessssssiiiooonnnooonnnttthhheeesssuuurrrfffaaaccceeeooofffcccaaannnccceeerrrccceeellllllsss······················14

222...IIInnnvvviiitttrrroootttrrraaannnsssddduuuccctttiiiooonnnmmmeeedddiiiaaattteeedddbbbyyyaaadddeeennnooovvviiirrraaalllvvveeeccctttooorrraaannndddaaadddeeennnooovvviiirrruuusss---llliiipppooosssooommmeeecccooommmpppllleeexxxeeesss···········································16

333...CCCyyytttoootttoooxxxiiiccciiitttyyyooofffcccaaatttiiiooonnniiicccllliiipppooosssooommmeeessscccooommmpppllleeexxxeeedddtttoooAAAddd---vvveeeccctttooorrrsss····21444...IIInnnvvviiitttrrroootttrrraaannnsssfffeeeccctttiiiooonnnbbbyyycccaaatttiiiooonnniiicccllliiipppooosssooommmeeesssaaannndddtttrrraaannnsssddduuuccctttiiiooonnnbbbyyyAAAddd---llliiipppooosssooommmeeecccooommmpppllleeexxxeeesss····················································23

- V -

555...IIInnnvvviiitttrrrooogggeeennneeetttrrraaannnsssddduuuccctttiiiooonnnmmmeeedddiiiaaattteeedddbbbyyyAAAddd---llliiipppooosssooommmeeecccooommmpppllleeexxxeeesssiiinnnvvvaaarrriiieeedddccceeellllllllliiinnneeesss···························································26

666...IIInnnvvviiivvvoootttrrraaannnsssddduuuccctttiiiooonnnbbbyyyAAAddd---vvveeeccctttooorrrsssaaannndddAAAddd---llliiipppooosssooommmeeecccooommmpppllleeexxxeeesss·····················································································29

ⅣⅣⅣ...DDDIIISSSCCCUUUSSSSSSIIIOOONNN ·································································35ⅤⅤⅤ...RRREEEFFFEEERRREEENNNCCCEEESSS ·······························································39국국국문문문 요요요약약약 ··············································································44

- VI -

LLLIIISSSTTT OOOFFFFFFIIIGGGUUURRREEESSS

FFFiiiggguuurrreee111...GGGeeennnooommmiiicccssstttrrruuuccctttuuurrreeeooofffAAAddd---CCCMMMVVV---GGGFFFPPP(((rrreeepppllliiicccaaatttiiiooonnndddeeefffeeeccctttiiivvveee---aaadddeeennnooovvviiirrruuussstttyyypppeee555)))·························································10

FFFiiigggrrruuueee222...DDDeeettteeeccctttiiiooonnn ooofffCCCAAARRR eeexxxppprrreeessssssiiiooonnn iiinnn vvvaaarrriiieeeddd cccaaannnccceeerrrccceeellllllllliiinnneeesssbbbyyyRRRTTT---PPPCCCRRR·····································································15

FFFiiiggguuurrreee 333...GGGFFFPPP eeexxxppprrreeessssssiiiooonnn bbbyyy AAAddd---vvveeeccctttooorrrsss iiinnn HHHeeeLLLaaa aaannnddd BBB111666BBBLLL666 CCCeeellllllsss···························································································································································································································18

FFFiiiggguuurrreee444... EEEffffffeeeccctttsssooofffaaammmooouuunnntttaaannndddcccooommmpppooosssiiitttiiiooonnnooofffcccaaatttiiiooonnniiicccllliiipppooosssooommmeeessscccooommmpppllleeexxxeeedddtttoooAAAddd---vvveeeccctttooorrrsssooonnnaaadddeeennnooovvviiirrraaallltttrrraaannnsssddduuuccctttiiiooonnniiinnnHHHeeeLLLaaaaaannndddBBB111666BBBLLL666ccceeellllllsss····················································································································································19

FFFiiiggguuurrreee 555...CCCyyytttoootttoooxxxiiiccciiitttyyy ooofffvvvaaarrriiiooouuusss cccaaatttiiiooonnniiiccc llliiipppooosssooommmeeesss cccooommmpppllleeexxxeeeddd wwwiiittthhhAAAddd---vvveeeccctttooorrrsss············································································22

FFFiiiggguuurrreee666...IIInnnvvviiitttrrroootttrrraaannnsssfffeeeccctttiiiooonnnbbbyyycccaaatttiiiooonnniiicccllliiipppooosssooommmeeesssaaannndddtttrrraaannnsssddduuuccctttiiiooonnnbbbyyyAAAddd---llliiipppooosssooommmeeecccooommmpppllleeexxxeeesssiiinnnHHHeeeLLLaaaaaannndddBBB111666BBBLLL666ccceeellllllsss···············24

FFFiiiggguuurrreee 777... IIInnn vvviiitttrrrooo tttrrraaannnsssddduuuccctttiiiooonnn bbbyyy AAAddd---vvveeeccctttooorrrsss aaannnddd AAAddd---llliiipppooosssooommmeeecccooommmpppllleeexxxeeesssiiinnnvvvaaarrriiiooouuussstttyyypppeeesssooofffcccaaannnccceeerrrccceeellllllsss······················28

- VII -

FFFiiiggguuurrreee 888...IIInnn vvviiivvvooo tttrrraaannnsssddduuuccctttiiiooonnn bbbyyy AAAddd---CCCMMMVVV---LLLuuuccc aaannnddd AAAddd---CCCMMMVVV---LLLuuuccccccooommmpppllleeexxxeeeddd wwwiiittthhh cccaaatttiiiooonnniiiccc llliiipppooosssooommmeeesss iiinnn aaa BBB111666BBBLLL666 mmmooouuussseeemmmooodddeeelll·······································································31

FFFiiiggguuurrreee999...IIInnnvvviiivvvoootttrrraaannnsssgggeeennneeeeeexxxppprrreeessssssiiiooonnnbbbyyyAAAddd---CCCMMMVVV---LLLuuucccaaannndddAAAddd---CCCMMMVVV---LLLuuuccc cccooommmpppllleeexxxeeeddd wwwiiittthhh vvvaaarrriiiooouuusss fffooorrrmmmuuulllaaatttiiiooonnnsss ooofff cccaaatttiiiooonnniiicccllliiipppooosssooommmeeesssiiinnn mmmaaajjjooorrrooorrrgggaaannnsssooofffmmmiiiccceeecccaaarrrrrryyyiiinnnggg BBB111666BBBLLL666tttuuummmooorrrsss·················································································32

FFFiiiggguuurrreee 111000...IIInnn vvviiivvvooo tttrrraaannnsssddduuuccctttiiiooonnn bbbyyy AAAddd---CCCMMMVVV---LLLuuuccc aaannnddd AAAddd---CCCMMMVVV---LLLuuuccccccooommmpppllleeexxxeeeddd wwwiiittthhh cccaaatttiiiooonnniiicccllliiipppooosssooommmeeesssiiinnn aaaHHHeeeLLLaaammmooouuussseeemmmooodddeeelll················································································33

FFFiiiggguuurrreee 111111... IIInnn vvviiivvvooo tttrrraaannnsssgggeeennneee eeexxxppprrreeessssssiiiooonnn bbbyyy AAAddd---CCCMMMVVV---LLLuuuccc aaannndddDDDMMMKKKEEE///CCChhhooolll///GGGaaalll---CCCeeerrr llliiipppooosssooommmeeesss cccooommmpppllleeexxxeeeddd wwwiiittthhhAAAddd---CCCMMMVVV---LLLuuuccc iiinnn mmmaaajjjooorrr ooogggrrraaannnsss ooofff mmmiiiccceee cccaaarrrrrryyyiiinnnggg HHHeeeLLLaaatttuuummmooorrrsss···································································34

- VIII -

LLLIIISSSTTT OOOFFFTTTAAABBBLLLEEESSS

TTTaaabbbllleeeⅠⅠⅠ...CCCooommmpppooosssiiitttiiiooonnnooofffcccaaatttiiiooonnniiicccllliiipppooosssooommmeeesss·····································6

TTTaaabbbllleeeⅡⅡⅡ...PPPrrriiimmmeeerrrsssfffooorrrRRRTTT---PPPCCCRRRaaannndddttthhheeeiiirrrPPPCCCRRRppprrroooddduuuccctttsssiiizzzeeesss···············8

- IX -

AAABBBBBBRRREEEVVVIIIAAATTTIIIOOONNNSSS

AAAddd:::aaadddeeennnooovvviiirrruuusssCCCAAARRR:::cccoooxxxsssaaaccckkkiiieeeaaadddeeennnooovvviiirrruuusssrrreeeccceeeppptttooorrrCCChhhooolll:::ccchhhooollleeesssttteeerrrooolllCCCMMMVVV:::cccyyytttooommmeeegggaaalllooovvviiirrruuusssDDDMMMKKKEEE:::OOO,,,OOO’’’---dddiiimmmyyyrrriiissstttyyylll---gggllluuutttaaammmyyylll---lllyyysssiiinnneeeFFFAAACCCSSS::: fffllluuuooorrreeesssccceeennnccceee---aaaccctttiiivvvaaattteeedddccceeellllllsssooorrrttteeerrrGGGaaalll---CCCeeerrr:::DDD---gggaaalllaaaccctttooosssyyylll---ββββ111---111’’’ccceeerrraaammmiiidddeeeGGGFFFPPP:::gggrrreeeeeennnfffllluuuooorrreeesssccceeennnccceeeppprrrooottteeeiiinnnLLLuuuccc:::llluuuccciiifffeeerrraaassseeeMMMCCCPPPSSS:::mmmooonnnooonnnuuucccllleeeaaarrrccceeellllllppphhhaaagggooocccyyytttiiicccsssyyysssttteeemmmMMMOOOIII:::mmmuuullltttiiipppllliiiccciiitttyyyooofffiiinnnfffeeeccctttiiiooonnnMMMTTTTTT:::333---(((444,,,555---dddiiimmmeeettthhhyyylllttthhhiiiaaazzzooolll---222yyylll)))---222,,,555---dddiiippphhheeennnyyylll---222HHH---ttteeetttrrraaazzzooollliiiuuummm bbbrrrooommmiiidddeeePPPBBBSSS:::ppphhhooosssppphhhaaattteee---bbbuuuffffffeeerrreeedddsssaaallliiinnneeePPPFFFUUU:::ppplllaaaqqquuueeefffooorrrmmmiiinnnggguuunnniiitttRRRLLLUUU:::rrreeelllaaatttiiivvveeellliiiggghhhtttuuunnniiittt

- X -

EEEffffffeeeccctttsssooofffcccaaatttiiiooonnniiicccllliiipppooosssooommmeeesssooonnniiinnnvvviiitttrrroooaaannndddiiinnnvvviiivvvoootttrrraaannnsssddduuuccctttiiiooonnnmmmeeedddiiiaaattteeedddbbbyyyaaadddeeennnooovvviiirrraaalll

vvveeeccctttooorrrsss

AAABBBSSSTTTRRRAAACCCTTT

Adenoviralvectorshavebeenwidelyusedingenetherapytrialsbecauseoftheirefficiencyingenetransfer.However,theirapplicationislimitedbytheirrequirementforcancercellstoexpressappropriatereceptorssuchascoxsackieadenovirusreceptor(CAR)invitroandby non-specificviraluptakeintheliver.To overcome both limits,in this study adenoviral vectors werecomplexedwithcationicliposomes,anon-viralgenecarriernon-specifictocelltypesand then tested in varied cancercelllinesand micecarrying tumormodels.Adenoviralvectors encoding green fluorescence protein (GFP) orluciferase (Luc)were mixed with O,O'-dimyristyl-glutamyl-lisine (DMKE),DMKE/cholesterol(Chol),orDMKE/Chol/D-galactosyl-β1-1’ceramide(Gal-Cer)liposomes to form adenovirus(Ad)-liposome complexes. Cancer cell linesexpressing CAR were effectively infected by adenoviralvectors withoutcationic liposomes,which was proved by fluorescence-activated cellsorter(FACS)analysisofGFPexpression.AdenoviraltransductiontoCAR-deficientB16BL6 and MCF-7 cancer cells was significantly enhanced by cationicliposomecomplexationtoadenoviralvectors.Meanwhile,thecationicliposomes

- XII -

variably affected in vitro adenoviralgene transduction to CAR-expressingHeLa,HepG2,andSNU739cells.Invivoadenoviraltransductionofluciferasegenewasimagedbyluminometer(IVISTM).Inordertoevaluatebiodistributionofadenoviralvectors,adenovirus or Ad-DMKE liposome complexes wereintravenously administered to C57BL/6 mice bearing CAR-deficientB16BL6mousemelanomacells.TheadenoviruscomplexedwiththeDMKE liposomesexhibited200-foldlowerhepatictransductionthanadenovirusalone.Meanwhile,geneexpressionintumortissueswasinsignificantlyaffectedbythecationicliposomes.The DMKE/Choland DMKE/Chol/Gal-Cerliposomes were moreeffectiveinreductionofadenoviralhepatotropism thantheDMKEliposomesinthesamemousemodel.AdenovirusandtheAd-liposomecomplexeswerealsoadministered to mice carrying CAR-positive tumors. When systemicallyadministered to BALB/c nu/nu mice carrying CAR-positive HeLa cells,Ad-vectors were readily accumulated in the mononuclear cellphagocyticsystem (MCPS)intheliverandspleen.However,adenoviruscomplexedwithDMKE/Chol/Gal-Cerliposomesexhibitednotransgeneexpressionintheliverandspleen.ThisstudysuggeststhatthecationicliposomescomplexedwithAd-vectorsenhanceinvitroadenoviraltransductiontoCAR-deficientcellsaswellasreducehepatotropism ofAd-vectors.

Keyword:adenovirus,genetherapy,cationicliposomes,coxsackieadenovirus

receptor(CAR),MCPS(mononuclearcellphagocyticsystem)

- 1 -

ⅠⅠⅠ...IIINNNTTTRRROOODDDUUUCCCTTTIIIOOONNNUseofrecombinanthumanadenoviralvectorscontinuestoshow increasing

promiseasgenetherapyvehicles,expeciallyforcancergenetherapy,duetoseveralimportantattributes,suchasitsexcellentgenetransferefficiencytonumerousdividingandnon-dividingcelltargets.Inaddition,adenoviralvectors(Ad-vectors)arerarely linked to any severediseasesin immunocompetenthumans,providing arationaleforfurtherdevelopmentofthesevehicles(1).Entryofadenoviralvectorsintomosttargetcellsismediatedbytheinitialbinding oftheadenoviralfibertothecoxsackieadenovirusreceptor(CAR)onthecellmembrane(2).CAR expressionandefficiencyofadenoviraltransgeneexpression have been correlated in a number ofstudies,suggesting thatadenoviralbinding andentryintotargetcellsisacriticalstepinachievingsuccessfuladenoviralgene expression (3).However,one of the hurdlesconfrontinggenetransferbyadenoviralvectorsistheirinefficienttransductiontotargetcellslackingsufficientexpressionofCAR;suchcellsincludemanyadvanced cancercells,skeletalmusclecells,smooth musclecells,peripheralbloodcells,hematopoieticstem cells,dendriticcells,andsoon(4).A highdoseofvectorsisrequiredtoachieveefficientgenetransfertothesecelltypes.This in turn increases unwanted side effects,such as vector-associatedimmunogenictoxicities(4).Another hurdle confronting Ad-vector-mediated gene transfer is their

nonspecificdistributionintissuesafterinvivogenetransferbecauseoftherelativelybroadexpressionofCAR.Thispropertyimposesanincreasedriskoftoxicity due to vectordissemination to non-targeting cells (4).Also,largenumbersofvirusarestillremovedbythemononuclearcellphagocyticsystem(MCPS)oftheliveraftersystemicadministration,resulting in reduction ofvectorplasmacirculationtime(5-8).Therefore,afterintravenousinjectionto

- 2 -

mice,adenoviralvectorsareinvolvedinaccumulationinmouseliver.Based on a clear understanding of Ad-vector cell recognition, the

developmentofCAR-independentAd-vectors has rationally focused on thefiberprotein,theprimarydeterminantofAd-vectortropism (1).Strategiestoeliminate hepatotropism,based on modification of particular viral capsidproteinssuchasfiberandpentonbase(9)havebeenreported.Instead of modification of adenoviral vector, surface modification of

Ad-vectorswithnon-viralgene-transferringsystemshasbeenconsideredasanotheroptionforreductionofadenoviralhepatotropism.Complexingadenoviralvectorswithcationicliposomesorpolymershasbeenshowntobeeffectiveinincreasingadenoviraltransgeneexpressionbyfacilitatingadenovirusbindingtothe cellsurface via rather non-specific charge interaction,expecially onCAR-deficient celllines (10-13).Ad-liposome complexes also resulted inreducedimmunogenicityofadenovirus(14).Numerousstudiesonnon-viralgenetransferutilizingliposomalvectorshave

shown thatlipid composition is importantin determining the efficacy ofliposome-mediatedgenetransfer(15).Thisislikelytobeduetoeffectsonthephysicalstability,intracellularuptake,endosomalrelease,andnuclearlocalizationofliposome-plasmidDNA complexes,allofwhichaffecttheultimatetransgeneexpression(16).Thecomplexesbetweenliposomesandadenoviralvectorsareknowntoform cationicliposomescoatingonthenegativelychargedsurfaceofadenovirus particles by electronic interactions similar to those inliposome-plasmidDNA complexes(16).Therefore,lipidcompositionsmay bealsoanimportantparameterdeterminingtransductionefficiencyofAd-liposomecomplexes.Consistent with these considerations, in this study the Ad-liposome

complexeswerepreparedwithDMKE (O,O’-dimyristyl-glutamyl-lisine)-basedliposomesandthenutilizedtoinfectvarioustypesofcancercellswithvaried

- 3 -

amountofCAR expression.TheAd-liposomecomplexes weresystemicallyadministeredtoananimalmodeltoevaluatetheeffectofcationicliposomesonbiodistributionandinvivotransductionefficacyofAd-vectors.Thisstudywillprovide importantinformation regarding how to resolve the two hurdles,inefficienttransductiontoCAR-deficientcellsandhepatotropism ofAd-vectors.

- 4 -

ⅡⅡⅡ...MMMAAATTTEEERRRIIIAAALLLSSSAAANNNDDD MMMEEETTTHHHOOODDDSSS111...MMMaaattteeerrriiiaaalllsss

D-galactosyl-β1-1'-ceramide was purchased from Avanti Polar Lipid(Alabaster,USA).Cholesterolwaspurchased from Sigma(St.Louis,USA).DMKE werechemically synthesized by Dr.Doo-Ok Jang (DepartmentofChemistry,YonseiUniversity,Korea).Accupower® RT premixwaspurchasedfrom Bioneer(Daejeon,Korea).C57BL/6miceand BALB/cnu/numicewereobtainedfrom OrientBio(Sungnam,Korea).TherecombinantAd-CMV-GFP(replicationdefectiveadenovirustype5)waskindlyprovidedbyDr.Chae-OkYun (Department of Medicine, Yonsei University, Seoul, Korea). TherecombinantAd-CMV-LucwaskindlyprovidedbyDr.Tae-SubLee(KoreaCancerCenterHospital,Seoul,Korea).Theviralstockwerekeptfrozenat-80℃ untiluse.The plasmid,pEGFP-N1 encoding green fluorescentproteindrivenbytheCMV promoterwaspurchasedfrom ClontechLaboratories(PaloAlto,USA).

- 5 -

222...CCCeeellllllllliiinnneeesssaaannndddccceeellllllcccuuullltttuuurrreee HumanhepatocellularcarcinomaSNU-739waspurchasedfrom KoreanCellLine Bank (Seoul,Korea).Human breastadenocarcinoma MCF-7,humancervicaladenocarcinoma HeLa,human hepatocellularcarcinoma HepG2 andmousemelanomaB16BL6werepurchased from theAmerican TypeCultureCollection (Manassas,USA).MCF-7 and HepG2 cells were maintained asmonolayerculturesin DMEM (Gibco,Carlsbad,USA).SNU-739cellswerecultureedinRPMI1640,B16BL6andHeLacellswereculturedinMEM (Gibco,Carlsbad,USA)medium.Theallmediaweresupplemented with 10% fetalbovine serum,100 units/㎖ penicillin and 100 ㎍/㎖ streptomycin in ahumidifiedatmosphereof95% airand5% CO2at37℃.

333...PPPrrreeepppaaarrraaatttiiiooonnnooofffcccaaatttiiiooonnniiicccllliiipppooosssooommmeeesssDMKE and cholesterol(Chol)were mixed ata molar ratio of60:40.

Gal-liposomes consisting ofDMKE,Choland D-galactosyl-β1-1'ceramide(Gal-Cer)weremixedatamolarratioof60:35:5(Table1).Thelipids(DMKE,Chol,andGal-Cer)weredissolvedinchloroform andmethanolmixture(2:1,v/v).Theorganicsolventwasevaporatedunderastream ofN2gas.Vacuumdesiccationfor2hoursensuredremovaloftheresidualorganicsolvent.Then,dried lipidfilm formedwashydrated in 1㎖ distilledwaterwith vigorousvortexing.Afterhydration,thedispersionwassonicated.Eachsuspensionwasextruded through polycarbonatemembranesofa100nm poresizeat60℃using an extruder (Avanti Polar Lipids, Alabaster, USA). The lipidconcentrationwasadjustedto1㎎/㎖.Allliposomeswerestoredat4℃ untiluse.

- 6 -

TTTaaabbbllleee111...CCCooommmpppooosssiiitttiiiooonnnooofffcccaaatttiiiooonnniiicccllliiipppooosssooommmeeesss

Compositions

Liposomes DMKE Cholesterol Gal-Cer

DMKE (100 molar ratio) 100% 0% 0%

DMKE/Chol (60:40 molar ratio) 60% 40% 0%

DMKE/Chol/Gal(60:35:5 molar ratio) 60% 35% 5%

- 7 -

444...RRRTTT---PPPCCCRRR aaannnaaalllyyysssiiisssfffooorrrCCCAAARRR eeexxxppprrreeessssssiiiooonnnooonnnttthhheeesssuuurrrfffaaaccceeeooofffcccaaannnccceeerrrccceeellllllsss Totalcellular RNA was extracted from cells.The cDNA againsttheextractedRNA wassynthesizedwitholigo(dT)primersandAccupower® RTpremix(Bioneer,Daejeon,Korea)accordingtothemanufacture'sinstructions,givingafinalvolumeof50㎕ ofcDNA preparation.ThePCR amplificationwascarriedoutwith2㎕ ofcDNA preparationandaspecificprimerpairofCAR (Table2).GAPDH wasusedasaninternalstandard(Table2).Twenty㎕ ofaPCR reactionsolutionwaspreparedusingAccupower® RT premix:1㎕ (10pmole)ofeachsenseandantisenseprimer,1.5㎕ ofcDNA sampleand16.5 ㎕ ofdistilled water.The mixture was submitted to 25 cycles ofamplification.In each cycle,denaturation was done at94℃ for 20 sec,annealing ofprimerstotargetcDNA wascarriedout61℃ for30sec,andextensionwasdoneat72℃ for1min,followedbyafinal10minelongationat72℃.The PCR products were visualized by ultravioletillumination afterelectrophoresisin1% TBEagarosegelandstainingwithethidium bromide.

- 8 -

TTTaaabbbllleee222...PPPrrriiimmmeeerrrsssfffooorrrRRRTTT---PPPCCCRRRaaannndddttthhheeeiiirrrPPPCCCRRRppprrroooddduuuccctttsssiiizzzeeesss

Name Type Sequence Product size

CAR

Sense 5’-GCCTTCAGGTCGCAGATGTTAC-3’

567 bpAnti-sense 5’-TCGCACCCATTCGACTTAGA-3’

GAPDH

Sense 5’-GTCAACGGATTTGGTCGTATT-3’

631 bpAnti-sense 5’-AGTCCTTCTGGGTGGCAGTGAT-3’

- 9 -

555...IIInnnvvviiitttrrroooaaadddeeennnooovvviiirrraaallltttrrraaannnsssddduuuccctttiiiooonnnTherecombinantadenoviralAd-CMV-GFPwasanE1andE3-deletedtype

5 adenoviralvector(Figure 1).The CMV represents the cytomegaloviruspromoterand arrowsshow theirdirection oftranscription.Thedeleted E1regionhadaGFPexpressioncassette.Adenoviralvectorswerepropagatedin293 celllines.Thevectorswerethen diluted to a titerof24.×1012 vp/㎖.Cancercells were seeded in 24-wellculture plates ata density of5×104

cells/wellandwereusedwhen70-80% confluent.Complexes ofAd-vectors and cationic liposomes were made by mixing

Ad-CMV-GFP andthepreparedliposomesin100㎕ ofserum freemedium,followed by incubation for30min atroom temperature.Cancercellswerewashedwithphosphate-bufferedsaline(PBS)andthepreparedAd-liposomecomplexeswerethen addedwith 400㎕ ofserum freemedium.After4hincubationinaCO2 incubatorat37℃,thecellswerewashedwithPBS toremovethecomplexes,and1㎖ offresh10% serum-containingmedium wasaddedtothecells.Thetransducedcellswerethenincubatedforanadditional48hbeforeassessingGFPexpression.

- 10 -

FFFiiiggguuurrreee 111...GGGeeennnooommmiiiccc ssstttrrruuuccctttuuurrreee ooofff AAAddd---CCCMMMVVV---GGGFFFPPP (((rrreeepppllliiicccaaatttiiiooonnn dddeeefffeeeccctttiiivvveeeaaadddeeennnooovvviiirrruuussstttyyypppeee555)))

- 11 -

666...AAAnnnaaalllyyysssiiisssooofffiiinnnvvviiitttrrroooGGGFFFPPPeeexxxppprrreeessssssiiiooonnnThetransducedcellswerewashedtwicewithPBSandfixedwith300㎕

of2% paraformaldehydefor5minat4℃.ThecellswerewashedtwicewithPBSagain,harvestedwithatrypsin-EDTA solution,andthenresuspendedin400㎕ medium with100㎕ FBS.Aftercentrifugationfor5minat1,500rpmat4℃,thesupernatantwasdiscardedandthefixedcellsresuspendedin500㎕ PBS.GFPexpressioninthesuspendedcellswasdirectlydeterminedinaFACS Caliburmachine(Beckton Dickinson,San Jose,USA).Ten thousandfluorescentevents per sample were acquired.Expression ofGFP in thetransduced cells was also viewed with a fluorescence microscopy andphotographed(magnification:×100).

777...CCCyyytttoootttoooxxxiiiccciiitttyyyaaassssssaaayyyooofffcccaaatttiiiooonnniiicccllliiipppooosssooommmeeesss CytotoxicityofcationicliposomescomplexedtoAd-vectorswasdeterminedby the MTT assay.Cancer cells were plated in 96-wellplates (1×104

cells/well)in0.1㎖ medium supplementedwith10% FBSandculturedfor24h. The cells were treated with the various concentrations of cationicliposomes-adenovirus complexes and then cultured for 24 h.Fity ㎕ of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)(Sigma,StLouis,USA)solution (1㎎/㎖)wasaddedtothecells,whichwerefurtherincubatedfor4h.AfterthemediacontainingMTT wereremoved,150㎕ ofDMSO wasaddedtosolubilizetheMTT-formazanproductinthecells.Theabsorbanceat540nm wasmeasured with amicroplatesreader(MolecularDevices,Sunnyvale,USA).

- 12 -

888...IIInnnvvviiivvvooogggeeennneeetttrrraaannnsssddduuuccctttiiiooonnnaaannndddaaannnaaalllyyysssiiisssoooffftttrrraaannnsssgggeeennneeeeeexxxppprrreeessssssiiiooonnnA HeLaxenograftmodelwaspreparedbysubcutaneousinoculationof5×106

HeLacellsin100㎕ ofmedium to5-week-oldfemalenudemice.A B16BL6xenograftmodelwas also prepared by subcutaneous inoculation of5×106

B16BL6cellsin100㎕ ofmedium to5-week-oldfemaleC57BL/6mice.Anindividualtumor-bearing mouse in groups was intravenously injected with3×108 pfu of Ad-CMV-Luc or Ad-CMV-Luc complexed with cationicliposomes (120 ㎍ oflipids)in a finalvolume of150 ㎕ of2.5% w/vglucose-saline.At6hand24hpostinjection,themicewereanesthetizedwithisoflurane.Then,thesamemicewereintraperitoneallyinjectedwithD-luciferinsubstratedissolvedinsterilePBS(30㎎/㎖)andimagedwithaluminometer.Theanimalswereplacedinalight-tightchamberandagray-scalereference

image was obtained underthe low-levelillumination.The florescence andphotonsemittedfrom themice,transmittingthroughthetissues,werecollectedwith a cooled charged-coupled device (CCD) camera (Xenogen,Alameda,USA).Theacquisitiontimewasrangedfor3min.Theresultswereanalyzedusing Living Image® software (Xenogen, Alameda, USA). The bodytemperaturewasmaintainedthroughoutimagingusinga37℃ platform inthechamber.Afteradministrationofluciferinsubstrate,themicewereimagedforaperiodoftimerangingfrom 1secto3min,dependingonsignalintensity.Raw valuesofluciferaseexpressionwererecodedasphotonsoflightemittedpersecond.Afterphotographed,theliver,lungs,spleen,hearts,kidney,intestine,stomach

andtumortissueswereharvested.Theharvestedorganswerewashedwithcoldsalinetwiceandhomogenizedinalysisbuffer(0.1M Tris-HCl,2mMEDTA,and 0.1% Triton X-100,pH 7.8) using a Heidolph tissue tearor

- 13 -

(ScientificSupport,Hayward,USA).Thetissuemixtureswerecentrifugedat14,000 rpm for 2 min at4℃.Ten ㎕ ofsupernatantwas subjected tomeasurementofluciferase activity in a luminometerwith a luciferase kit(PropmegaBiosciences,SanLuisObispo,USA).Tocalculatetherelativelightunit(RLU),theproteinconcentrationofthesamplewasalsomeasuredwiththeDCproteinassaykit(Bio-Rad,Hercules,USA).

- 14 -

ⅢⅢⅢ...RRREEESSSUUULLLTTTSSS

111...CCCAAARRReeexxxppprrreeessssssiiiooonnnooonnnttthhheeesssuuurrrfffaaaccceeeooofffcccaaannnccceeerrrccceeellllllssscDNA wassynthesizedfrom totalRNA extractedfrom variouscelllines

byreversetranscriptase.cDNA sequencesofCARandGAPDH wereamplifiedwiththedesignatedprimeroligonucleotidesmentionedearlier(Table2).ThesizeofeachPCR productwasasfollow;CAR,567bp;GAPDH,631bp.ThePCR productswereelectrophoresedin1% TBEagarosegelandvisualizedbyultravioletillunminationafterstainingwithethidium bromide.CARmRNA levelwashighinHeLa,HepG2andSNU-739celllines(Figure2).Incontrast,thereceptorwasnotexpressedinB16BL6andMCF-7(Figure2).Hence,HeLaforCAR-expressing celllineand B16BL6forCAR-deficientcelllinewereselectedforfurtherinvitroandinvivotrasnductionstudieswithAd-vectorandAd-liposomecomplexesascancermodels.

- 15 -

FFFiiiggguuurrreee222...DDDeeettteeeccctttiiiooonnn ooofffCCCAAARRR eeexxxppprrreeessssssiiiooonnn iiinnn vvvaaarrriiieeeddd cccaaannnccceeerrrccceeellllllllliiinnneeesssbbbyyyRRRTTT---PPPCCCRRR...cDNAsofCARandGAPDH (acontrolofsynthesizedRNA quality)wereamplifiedwiththeirspecificprimers.PCRproductswereseparatedon1%agarosegelsandstainedwithethidium bromide.Lane1,100bpmarker;lane2,HeLa;lane3,HepG2;lane4,SNU-739;lane5,B16BL6;lane6,MCF-7.

- 16 -

222...IIInnnvvviiitttrrroootttrrraaannnsssddduuuccctttiiiooonnnmmmeeedddiiiaaattteeedddbbbyyyaaadddeeennnooovvviiirrraaalllvvveeeccctttooorrraaannndddaaadddeeennnooovvviiirrruuusss---llliiipppooosssooommmeeecccooommmpppllleeexxxeeesssInordertocomparetransductionefficiencyofAd-vectorsandAd-liposome

complexesinCAR-expressingandCAR-deficientcancercells,HeLacellsandB16BL6cellsweretransducedwiththetwogene-transferringsystems.Firstofall,the optimalMOI(multiplicity ofinfection)ofadenoviralvectors wasdetermined for effective gene transduction.After 4 h transduction andadditional48hincubation,GFPexpressioninthetransducedcellswasviewedwith afluorescencemicroscopy andby theFACS analysis.Addition ofthehigheramountofAd-vectors exhibited the more efficientGFP expression(Figure3).AccordingtotheresultofGFP expressioninHeLacells(Figure3A),nearly allcells weretransduced in thepresence ofthe 100 MOIofAd-vectors.Meanwhile,CAR-deficient B16BL6 cells were not effectivelytransducedwithAd-vectors(Figure3B).TheseresultsclearlyshowedthatGFP expression mediatedby Ad-vectorswasdependentupon expression ofCAR on the cellsurface and 100 MOI(5×106 pfu)ofAd-vectorwas anoptimaltiterfortransductiontoCAR-expressingcancercells.Threedifferenttypesofcationicliposomeswerepreparedasdescribedin

Table1:DMKE liposomes,DMKE/Cholliposomes,and DMKE/Chol/Gal-Cerliposomes.Variedamountofthesecationicliposomeswerecomplexedwithafixed amountofAd-vector(100MOI).HeLa cellsand B16BL6cellsweretransduced by Ad-vectors and the prepared Ad-liposome complexes toinvestigateeffectsofcationicliposomesonadenoviralgenetransductionandtodeterminetheoptimalratioofliposomeandAd-vectorsforefficientinvitroandinvivotransduction.LiposomecomplexationlittleaffectedthetransductionefficiencyofAd-vectorsinHeLacells(Figure4A).AccordingtotheFACSanalysis,theGFPexpressionmediatedbyAd-vectorswasslightlyhigherthan

- 17 -

thatby Ad-liposomecomplexes.Among thecationicliposomes,transductioninterferencebyDMKE/Cholliposomeswasslightlyhigherthanthosebytheotherformulations.In contrast,adenoviraltransduction in CAR-deficientB16BL6 cells was

enhancedbycomplexationwithcationicliposomes.Especially,theadenoviruscomplexedwiththeDMKE liposomes(2㎍ lipid/㎖)wasabletotransduce26-fold highernumberofcellsthan theadenovirusonly.ThehighestGFPexpressionwasobtainedinthepresenceof0.5∼2㎍/㎖ ofDMKE liposomes.The highest GFP expression with the DMKE/Chol liposomes and theDMKE/Chol/Gal-Cerliposomeswasseenat0.5and4lipid㎍/㎖,respectively.According totransduction resultsin both celllines,theDMKE liposomes

(0.5～1㎍/㎖)insignificantlyaffecttheadenoviraltransductioninCAR-positivecells,butsignificantlyenhancedthetransductioninCAR-negativecells.

- 18 -

FFFiiiggguuurrreee333...GGGFFFPPP eeexxxppprrreeessssssiiiooonnn bbbyyy AAAddd---vvveeeccctttooorrrsssiiinnn HHHeeeLLLaaa aaannnddd BBB111666BBBLLL666 CCCeeellllllsss...VariousMOIsofAd-vectorswereaddedtoHeLacells(A)andB16BL6(B),transducedfor4h,andincubatedforadditional48h.GFPexpressionofthecellswasexaminedunderalightmicroscopy(upperlow)andafluorescencemicroscopy(lowerlow).

000MMMOOOIII 111000MMMOOOIII 111000000MMMOOOIII 222000000MMMOOOIII 444000000MMMOOOIII

(((AAA)))

(((BBB)))

000MMMOOOIII 111000MMMOOOIII 111000000MMMOOOIII 222000000MMMOOOIII 444000000MMMOOOIII

- 19 -

(A) (A) (A) (A) HeLaHeLaHeLaHeLa

0000

20202020

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60606060

80808080

100100100100

UntransducedUntransducedUntransducedUntransduced

cellscellscellscells

Ad onlyAd onlyAd onlyAd only 0.50.50.50.5 1111 2222 4444

Concentration of liposomes added Concentration of liposomes added Concentration of liposomes added Concentration of liposomes added ((((㎍㎍㎍㎍////㎖㎖㎖㎖))))

GF

P e

xpressin

g c

ell

s (

%)

GF

P e

xpressin

g c

ell

s (

%)

GF

P e

xpressin

g c

ell

s (

%)

GF

P e

xpressin

g c

ell

s (

%)

DMKEDMKEDMKEDMKE

DMKE/CholDMKE/CholDMKE/CholDMKE/Chol

DMKE/Chol/Gal-CerDMKE/Chol/Gal-CerDMKE/Chol/Gal-CerDMKE/Chol/Gal-Cer

- 20 -

(B) (B) (B) (B) B16BL6B16BL6B16BL6B16BL6

0000

20202020

40404040

60606060

80808080

100100100100

UntransducedUntransducedUntransducedUntransduced

cellscellscellscells

Ad onlyAd onlyAd onlyAd only 0.50.50.50.5 1111 2222 4444

Concentration of liposomes added Concentration of liposomes added Concentration of liposomes added Concentration of liposomes added ((((㎍㎍㎍㎍////㎖㎖㎖㎖))))

GFP

expressin

g c

ell

s (

%)

GFP

expressin

g c

ell

s (

%)

GFP

expressin

g c

ell

s (

%)

GFP

expressin

g c

ell

s (

%)

DMKEDMKEDMKEDMKE

DMKE/CholDMKE/CholDMKE/CholDMKE/Chol

DMKE/Chol/Gal-CerDMKE/Chol/Gal-CerDMKE/Chol/Gal-CerDMKE/Chol/Gal-Cer

FFFiiiggguuurrreee 444...EEEffffffeeeccctttsss ooofff aaammmooouuunnnttt aaannnddd cccooommmpppooosssiiitttiiiooonnn ooofff cccaaatttiiiooonnniiiccc llliiipppooosssooommmeeessscccooommmpppllleeexxxeeeddd tttooo AAAddd---vvveeeccctttooorrrsss ooonnn aaadddeeennnooovvviiirrraaalll tttrrraaannnsssddduuuccctttiiiooonnn iiinnn HHHeeeLLLaaa aaannndddBBB111666BBBLLL666ccceeellllllsss...NumbersofHeLa(A)andB16BL6(B)cellsexpressingGFPwerecountedbyFACS analysis48hposttransductionwithAd-vectorsorAd-liposomecomplexes.

- 21 -

333...CCCyyytttoootttoooxxxiiiccciiitttyyyooofff cccaaatttiiiooonnniiicccllliiipppooosssooommmeeessscccooommmpppllleeexxxeeedddtttoooAAAddd---vvveeeccctttooorrrsss B16BL6cellswereincubatedin thepresenceofvariedconcentrationsofDMKE,DMKE/Chol,andDMKE/Chol/Gal-Cerliposomestomeasuretheircelltoxicities.Asdescribedelsewhere,thecationicliposomeswerecytotoxicattherelatively higherconcentration(>20㎍ lipid/㎖)(Figure5).However,attherangeoftheoptimizedlipidconcentrationforinvitrotransduction(

- 22 -

0000

20202020

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60606060

80808080

100100100100

0000 10101010 20202020 40404040 80808080 100100100100

Concentration of liposomes Concentration of liposomes Concentration of liposomes Concentration of liposomes ((((㎍㎍㎍㎍////㎖㎖㎖㎖))))

Grow

th (

%)

Grow

th (

%)

Grow

th (

%)

Grow

th (

%)

DMKEDMKEDMKEDMKE

DMKE/CholDMKE/CholDMKE/CholDMKE/Chol

DMKE/Chol/Gal-CerDMKE/Chol/Gal-CerDMKE/Chol/Gal-CerDMKE/Chol/Gal-Cer

FFFiiiggguuurrreee 555...CCCyyytttoootttoooxxxiiiccciiitttyyy ooofffvvvaaarrriiiooouuusss cccaaatttiiiooonnniiiccc llliiipppooosssooommmeeesss cccooommmpppllleeexxxeeeddd wwwiiittthhhAAAddd---vvveeeccctttooorrrsss...B16BL6 cells seeded in 96-wellplates (1×104 cell/well)wereincubated with varied doses of liposomes pre-complexed with 1×106 pfuAd-CMV-GFPfor2h.CellviabilitywasmeasuredbytheMTT assay.

- 23 -

444... IIInnn vvviiitttrrrooo tttrrraaannnsssfffeeeccctttiiiooonnn bbbyyy cccaaatttiiiooonnniiiccc llliiipppooosssooommmeeesss aaannndddtttrrraaannnsssddduuuccctttiiiooonnnbbbyyyAAAddd---llliiipppooosssooommmeeecccooommmpppllleeexxxeeesssGFPexpressionmediatedbyliposometransfectionwascomparedwiththatbyAd-liposome complexes.After 4 h transfection or transduction and thenadditional24 h or 48 h incubation,GFP expression in HeLa cells wasmeasured by FACS analysis.Thecellsweretransfected with 1㎍ pDNAcomplexed6㎍ ofliposomes(1:6wtratio)ortransducedwith100MOIofAd-vectorcomplexed1㎍/㎖ ofliposomes.Generally GFP expression by lipidic transfection was lowerthan thatbyadenoviraltransduction (Figure6).Asshown previously (Figure4),DMKE,DMKE/Chol or DMKE/Chol/Gal complexation did not affect transductionefficiency ofAd-vectors in HeLa cells (91.0±2.57,91.5±1.94,90.4±0.90,and93.7±1.68%,respectively)(Figure 6A).However,adenoviraltransduction toB16BL6 cells was significantly elevated by complexation with cationicliposomes,speciallyDMKE liposomes(Figure6B).AdditionofcholesterolorgalactosylceramidetotheDMKEliposomesreducedthetransfectionefficiencyofthecationicliposomesandtransductionefficiencyofAd-liposomecomplexes.This resultclearly shows thatcationic liposomes can help Ad-vectors totransducetoCAR-deficientcells.

- 24 -

(((AAA)))HHHeeeLLLaaa

0000

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80808080

100100100100

Un tr an sf ec te d

N ak ed p DNA

DMK E

D MKE /Ch ol

D MK E/ Ch ol / Ga l- Ce r

Un tr an sd uc ed

Ad o nl y

Ad /DMKE

Ad /DMK E/ Ch ol

Ad /DMK E/ Ch ol / Ga l- Ce r

GFP

expre

ssin

g c

ell

s (

%)

GFP

expre

ssin

g c

ell

s (

%)

GFP

expre

ssin

g c

ell

s (

%)

GFP

expre

ssin

g c

ell

s (

%)

LLLiiipppooosssooommmeeesss AAAddd---llliiipppooosssooommmeeecccooommmpppllleeexxxeeesss

- 25 -

(((BBB)))BBB111666BBBLLL666

0000

20202020

40404040

60606060

80808080

100100100100

U nt ra ns fe ct ed

Na ke d pDNA

D MK E

D MKE /C ho l

D MKE /Ch ol / Ga l- Ce r

U nt ra ns du ce d

A d on ly

Ad /D M

K E

A d/ DMKE /C ho l

A d/ DMKE /Ch ol / Ga l- Ce r

GFP

expressin

g c

ell

s (

%)

GFP

expressin

g c

ell

s (

%)

GFP

expressin

g c

ell

s (

%)

GFP

expressin

g c

ell

s (

%)

LLLiiipppooosssooommmeeesss AAAddd---llliiipppooosssooommmeeecccooommmpppllleeexxxeeesss

FFFiiiggguuurrreee666...IIInnnvvviiitttrrroootttrrraaannnsssfffeeeccctttiiiooonnnbbbyyycccaaatttiiiooonnniiicccllliiipppooosssooommmeeesssaaannndddtttrrraaannnsssddduuuccctttiiiooonnnbbbyyyAAAddd---llliiipppooosssooommmeee cccooommmpppllleeexxxeeesss iiinnn HHHeeeLLLaaa aaannnddd BBB111666BBBLLL666 ccceeellllllsss...One ㎍ ofpDNAcomplexedto6㎍ ofDMKE,DMKE/Chol,orDMKE/Chol/Gal-Cerliposomesat1:6wtratio,or5×106 pfuofAd-vectorcomplexedto1㎍/㎖ ofDMKE,DMKE/Chol,orDMKE/Chol/Gal-CerliposomeswereaddedtoHeLa(A)andB16BL6cells(B).Thecellswereincubatedfor4handadditional24hfortransfection or 48 h for transduction.GFP expression in the cells wasmeasuredbyFACSanalysis.

- 26 -

555...IIInnnvvviiitttrrrooogggeeennneeetttrrraaannnsssddduuuccctttiiiooonnnmmmeeedddiiiaaattteeedddbbbyyyAAAddd---llliiipppooosssooommmeeecccooommmpppllleeexxxeeesssiiinnnvvvaaarrriiieeedddccceeellllllllliiinnneeesssLiposome-complexedadenoviralvectorshaveshownparticularlyeffectivein

CAR-deficientcelllines (11,14).Thus,in this study,effects ofcationicliposome complexation on transduction efficiency of Ad-vectors wereinvestigated in various cancercells,CAR-positive orCAR-negative.Aftertransduction by Ad-liposome complexes,GFP expression in the cells wasmeasuredbyFACS analysis.Thecellsweretransducedunderanoptimizedconditionof5×106pfuAd-vectorcomplexedto1㎍/㎖ ofDMKE,DMKE/CholorDMKE/Chol/Gal-Cercationicliposomes.InCAR-deficientB16BL6andMCF-7celllines,approximately3% and11%

ofthe respective cells were transduced with Ad-vectors alone (Figure 7).DMKEorDMKE/Chol/Gal-Cerliposomecomplexationincreasedtheadenoviraltransduction by 5.5-fold and 27-fold in MCF-7 and B16BL6,respectively.Generally DMKE liposomes was the most effective in enhancement ofadenoviraltransduction in CAR-deficientcells among the prepared cationicliposomes.On the whole,Ad-vectors were able to transduce the GFP gene to

CAR-expressing cells more effectively than CAR-deficientcells.Underthesametransductioncondition,thepercentagesofcellstransducedbyAd-vectorsalonewereapproximately25%,44%,and91% inHepG2,SNU-739,andHeLacells. As shown previously (Figure 4), cationic lipid complexation toAd-vectors little affected the adenoviraltransduction to HeLa cells,butnegatively affected thetransduction to HepG2cells.Interestingly,thesameformulationenhancedthetransductiontoSNU-739cell,oneofCAR-positivecells. ComplexationwiththeDMKE liposomesexhibitedapproximately21%,92%, and 96% of transduction in HepG2, HeLa, and SNU-739 cells,

- 27 -

respectively. Under the same experimental conditions, the DMKE/CholliposomesandDMKE/Chol/Gal-CerliposomesalsoexhibitedthesimilareffectwiththeDMKEliposomes.Theseresultsindicatethatcationicliposomecomplexationgenerallyenhances

adenoviralgene transferto CAR-deficientcells and affectadenoviralgenetransfertoCAR-expressing cellspositively ornegatively,depending on celltypes.

- 28 -

0000

20202020

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80808080

100100100100

120120120120

UntransducedUntransducedUntransducedUntransduced

cellscellscellscells

Ad onlyAd onlyAd onlyAd only DMKEDMKEDMKEDMKE DMKE/CholDMKE/CholDMKE/CholDMKE/Chol DMKE/Chol/Gal-DMKE/Chol/Gal-DMKE/Chol/Gal-DMKE/Chol/Gal-

CerCerCerCer

GF

P e

xpressin

g c

ell

s (

%)

GF

P e

xpressin

g c

ell

s (

%)

GF

P e

xpressin

g c

ell

s (

%)

GF

P e

xpressin

g c

ell

s (

%)

HepG2HepG2HepG2HepG2

MCF-7MCF-7MCF-7MCF-7

B16BL6B16BL6B16BL6B16BL6

HeLaHeLaHeLaHeLa

SNU-739SNU-739SNU-739SNU-739

FFFiiiggguuurrreee 777... IIInnn vvviiitttrrrooo tttrrraaannnsssddduuuccctttiiiooonnn bbbyyy AAAddd---vvveeeccctttooorrrsss aaannnddd AAAddd---llliiipppooosssooommmeeecccooommmpppllleeexxxeeesss iiinnn vvvaaarrriiiooouuusss tttyyypppeeesss ooofff cccaaannnccceeerrr ccceeellllllsss...Ad–vectors (5ⅹ106 pfu)complexedwith1㎍/㎖ ofDMKE,DMKE/CholandDMKE/Chol/Galliposomeswereaddedtovarioustypesofcancercells.Thecellswereincubatedfor4handthenadditional48h.GFPexpressioninthecellswasmeasuredbyFACSanalysis.

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666...IIInnnvvviiivvvoootttrrraaannnsssddduuuccctttiiiooonnnbbbyyyAAAddd---vvveeeccctttooorrrsssaaannndddAAAddd---llliiipppooosssooommmeeecccooommmpppllleeexxxeeesssInordertoverifyeffectofcationicliposomecomplexationoninvivogene

transduction activities ofadenoviralvectors,Ad-liposome complexes wereintravenouslyadministeredintoB16BL6tumor-bearingC57BL/6miceandHeLatumor-bearingBALB/cnc/ncnudemiceviatailvain. Themajororgansofthemononuclearcellphagocyticsystem (MCPS)includingtheliverandspleenwereexaminedforluciferasetransgeneexpressionfollowingtailvaininjectionof Ad-liposome complexes or Ad-vectors. The transduced mice wereintraperitoneallyinjectedwithD-luciferinsubstratedissolvedinsterilePBS6hand24hpostinjection,andthenimagedwithaluminometer.Thefluorescenceand photonsemitted from themice,transmitting through thetissues,werecollected with a cooled charge-coupled device (CCD) camera (Xenogen,Alameda,USA)As a result,in C57BL/6 mice carrying B16BL6 tumors,the adenoviral

vectorswerepredominantlylocalizedinlivertissues(Figure8).Meanwhile,themicetreatedwith threedifferenttypesofAd-liposomescomplexes(DMKE,DMKE/Chol,andDMKE/Chol/Gal-Cerliposomes)exhibitedsignificantlylowerluciferaseexpressionlevelsintheliverandspleenthantheonestreatedwithAd-vectors alone (Figure 8).The reduction ofhepatic and splenic geneexpression by cationicliposomecomplexation werenotdirectly related withenhancementoftransductionintheotherorgans.Onlytumortissuesexhibiteda slightly enhanced transgene expression by the Ad-DMKE liposomecomplexes(Figure9).TheDMKE/CholandDMKE/Chol/Gal-Cerliposomesnotonly significantly removedhepatictropism oftheadenoviralvectors,butalsoreducedtransductiontotumortissues(Figure9).InHeLatumor-bearingnc/ncnudemice,theadenoviralvectorswerealso

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predominantlylocalizedinlivertissues6h,butthetransgeneexpressionfadedawayin24hpostinjection(Figure10).Thereductionoftransgeneexpressionbycationicliposomecomplexationdidnotyieldenhancedtransductiontoanyotherorgansincludingtumors.Thetreated miceweresacrificed 24h postinjection and luciferasegene

expression in themajororganswasviewed with aluminometer.GenerallyAd-vectorsexhibitedhigherluciferaseexpressionininternalorgansincludingtheliverandspleen(Figure11).TheDMKE/Chol/Gal-Cerliposomesloweredthe hepatic transduction and transduction to otherorgans as well.Theseresultsindicatethathepatictransduction by Ad-vectorscan bereduced bycationicliposomecomplexation,butthecurrentformulationofthecomplexesisnotgoodenoughtoredistributetheresidualviralcomplexestootherorgans,speciallytotumortissues.

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AAA

mouse mouse mouse mouse 1 1 1 1 mouse mouse mouse mouse 2 2 2 2 mouse mouse mouse mouse 3 3 3 3 mouse mouse mouse mouse 4 4 4 4 mouse mouse mouse mouse 5 5 5 5 mouse mouse mouse mouse 6 6 6 6

666hhhpppooossstttiiinnnjjjeeeccctttiiiooonnn 222444hhhpppooossstttiiinnnjjjeeeccctttiiiooonnnFFFiiiggguuurrreee 888...IIInnn vvviiivvvooo tttrrraaannnsssddduuuccctttiiiooonnn bbbyyy AAAddd---CCCMMMVVV---LLLuuuccc aaannnddd AAAddd---CCCMMMVVV---LLLuuuccccccooommmpppllleeexxxeeeddd wwwiiittthhh cccaaatttiiiooonnniiiccc llliiipppooosssooommmeeesss iiinnn aaa BBB111666BBBLLL666 mmmooouuussseee mmmooodddeeelll...Ad-CMV-Luc (3×108 pfu)(A)and Ad-CMV-Luc complexed with DMKEliposomes(mouse1,mouse4),DMKE/Cholliposomes(mouse2,mouse5),orDMKE/Chol/Gal-Cer(mouse3,mouse6)(120㎍ lipid:3×108pfu)(B)wereintravenouslyinjectedintoB16BL6tumor-bearingC57BL/6miceviatailvein.ThemicewereintraperitoneallyinjectedwithD-luciferin(3mg/mouse)6hand24hpostinjection.Sixminutelater,thetreatedmicewereimagedbyaCCDIVISTM system.

BBBB

666hhhpppooossstttiiinnnjjjeeeccctttiiiooonnn 222444hhhpppooossstttiiinnnjjjeeeccctttiiiooonnn

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FFFiiiggguuurrreee 999... IIInnn vvviiivvvooo tttrrraaannnsssgggeeennneee eeexxxppprrreeessssssiiiooonnn bbbyyy AAAddd---CCCMMMVVV---LLLuuuccc aaannndddAAAddd---CCCMMMVVV---LLLuuuccccccooommmpppllleeexxxeeedddwwwiiittthhhvvvaaarrriiiooouuusssfffooorrrmmmuuulllaaatttiiiooonnnsssooofffcccaaatttiiiooonnniiicccllliiipppooosssooommmeeesssiiinnn mmmaaajjjooorrrooorrrgggaaannnsssooofffmmmiiiccceeecccaaarrrrrryyyiiinnnggg BBB111666BBBLLL666tttuuummmooorrrsss...Ad-CMV-Luc(3×108

pfu) or Ad-CMV-Luc with DMKE,DMKE/Choland DMKE/Chol/Gal-Cerliposomes(120㎍ lipid:3×108 pfu)wereintravenouslyinjectedintoB16BL6tumor-bearingC57BL/6miceviatailvein.Majororganswerecollected24hpostinjectionandtheluciferaseexpressionintheorganswascalculatedtoRLU ofluciferasepermilligram ofprotein.

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mmmooouuussseee111 mmmooouuussseee222 mmmooouuussseee333 mmmooouuussseee444666hhhpppooossstttiiinnnjjjeeeccctttiiiooonnn 222444hhhpppooossstttiiinnnjjjeeeccctttiiiooonnn

FFFiiiggguuurrreee 111000...IIInnn vvviiivvvooo tttrrraaannnsssddduuuccctttiiiooonnn bbbyyy AAAddd---CCCMMMVVV---LLLuuuccc aaannnddd AAAddd---CCCMMMVVV---LLLuuuccccccooommmpppllleeexxxeeedddwwwiiittthhhcccaaatttiiiooonnniiicccllliiipppooosssooommmeeesssiiinnnaaaHHHeeeLLLaaammmooouuussseeemmmooodddeeelll...Ad-CMV-Luc (3×108 pfu) (mouse 1, mouse 3) or DMKE/Chol/Gal-CerliposomescomplexedwithAd-CMV-Luc(120㎍ lipid:3×108pfu)(mouse2,mouse4)wereintravenouslyinjectedintoHeLatumor-bearingBALB/cnc/ncnude mice via tailvein.The mice were intraperitoneally injected withD-luciferin(3mg/mouse)6hand24hpostinjection.Sixminutelater,thetreatedmicewereimagedbyaCCDIVISTM system.

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FFFiiiggguuurrreee 111111... IIInnn vvviiivvvooo tttrrraaannnsssgggeeennneee eeexxxppprrreeessssssiiiooonnn bbbyyy AAAddd---CCCMMMVVV---LLLuuuccc aaannndddDDDMMMKKKEEE///CCChhhooolll///GGGaaalll---CCCeeerrrllliiipppooosssooommmeeessscccooommmpppllleeexxxeeeddd wwwiiittthhh AAAddd---CCCMMMVVV---LLLuuuccciiinnn mmmaaajjjooorrrooorrrgggaaannnsssooofffmmmiiiccceeecccaaarrrrrryyyiiinnnggg HHHeeeLLLaaatttuuummmooorrrsss...Ad-CMV-Luc(3×108 pfu)(A)orAd-CMV-LucwithDMKE/Chol/Gal-Cerliposomes(120㎍ lipid:3×108 pfu)(B) wereintravenouslyinjectedintoHeLatumor-bearingBALB/cnu/nunudemiceviatailvein.ThemicewereintraperitoneallyinjectedwithD-luciferin(3mg/mouse)24hpostinjection.Sixminutelater,thetreatedmicewereimagedbyaCCDIVISTM system.

Liver, Lung Spleen Liver, Lung Spleen Liver, Lung Spleen Liver, Lung Spleen KidneyKidneyKidneyKidney HeartHeartHeartHeart

Stomach Intestine HeLa tumorStomach Intestine HeLa tumorStomach Intestine HeLa tumorStomach Intestine HeLa tumor

(((AAA)))

(((BBB)))

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ⅣⅣⅣ...DDDIIISSSCCCUUUSSSSSSIIIOOONNNDespite a variety ofextensive efforts to improve human adenovirus

serotype5asatherapeuticagent,thisvectorexhibitsinnatedrawbacksofCAR dependency and hepatotropism.Of particular concern is that manyclinically importanttissues,including severalcancertypes,arerefractory toAd5infectionduetolow CARexpression.Indeed,down-regulationofCARhasbeenreportedforseveraltumortypes,includingglioma,ovariancancer,lungcancer,breastcancerandothers(17-19).Asaconsequenceoflimiting CARlevelsinmanyclinicallyrelevanttargetcells,ahighAd-vectordosageisoftenrequired forin vivo efficacy.Since over95% ofsystemically administeredadenoviralparticles are sequestered in the liver via hepatic macrophage(Kupffer cells) (20) and hepatocyte (21) uptake, therapeutically relevantadenoviraldosesoften resultsin vector-related livertoxicity (22-26).Thus,Ad-vectors exhibiting CAR-independency with low hepatotropism willbehelpfulformaximaltransductionoflow-CAR targetsatthelowestpossiblevectordose.Inthisstudy,Ad-vectorswerecomplexedwithcationicliposomes,resulting

inefficientCAR-independentinvitrotransductionandlow-hepatotropism whensystemically applied.According to the in vitro data with CAR-deficientB16BL6cells,complexationwiththeDMKE-basedcationicliposomesablatedCAR-dependenttransduction capability ofAd-vectors.Ad-vectorscompelxedwiththecationicliposomeexhibitedsignificantlyelevatedtransgeneexpressionin CAR-deficientB16BL6 cells.This clearly shows thatthe DMKE-basedcationicliposomesareabletoenhanceinvitroinfectivity ofAd-vectorstoCAR-deficient cancer cells.These results suggest that cationic liposomecomplexationprovidesCAR-independenttropism forAd-vectors.In contrast, in CAR-expressing cells, cationic liposome complexation

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exhibited avariety ofoutcomes,littleeffecton transduction toHeLacells,inhibitoryeffectontransductiontoHepG2cellsasdescribedelsewhere(16),andpositiveeffectontransductiontoSNU-739cells.Thismayimplythatinitialinteraction modes of the lipid-coated surface of Ad particles with theCAR-expressingsurfaceofcancercellsarevarieddependingonthenatureofcancercellsurface,suchasCAR density,electronegativity,andsoon.Thedifferent initial interaction modes may result in varied efficiency ofinternalizationofadenoviralparticlesintocytoplasm.Theamountandcompositionofcationicliposomesweremajorparameters

governing transduction efficiency of Ad-vectors complexed with cationicliposomes.The concentrations ofcationic liposomes affected the levelofadenoviral transduction efficiency. In CAR-expressing HeLa cells, whileaddition oftheDMKE liposomesupto4㎍/㎖ had noeffecton adenoviraltransduction,DMKE/CholandDMKE/Chol/Gal-Cerliposomesexhibitedsuddenreductionatthesameconcentration.InCAR-deficientB16BL6cells,theDMKEliposomesenhancedadenoviraltransductionstablyfrom 0.5to2㎍/㎖ oflipidconcentration.Meanwhile,theDMKE/CholandDMKE/Chol/Gal-Cerliposomesshowedmaximalenhancementofadenoviraltransductionat0.5and4㎍/㎖,respectively.Accordingtotheseresults,theDMKE-liposomeswerethemosteffective in mediating adenoviraltransduction in allcelllines tested.Theoptimizedconcentrationofliposomeswas1㎍/㎖ ofliposomesfor5×106pfuofadenovirus.IntravenousadministrationofAd-vectorsresultsinaccumulationintheliver,

spleen,heart,lung and kidneys ofmice,although theses tissues may notnecessarily be the highest in CAR expression (27-29).Instead,anatomicbarriers,thestructureofthevasculatureandthedegreeofbloodflow ineachorgan probably contributetotheirbiodistribution,inadditiontonon-specificviralparticleuptakebymacrophages.Thisistruewithregardtotheliverin

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particular, which sequesters the majority of systemically administeredadenoviralparticleviahepaticmacrophageuptake(20)aswellashepatocytetransduction(21).Thisprocessleadstocytokinerelease,inflammationandlivertoxicity (22,24,and 30).Thus,the nature of adenovirus-cellinteractionsdictating thefateofsystemically appliedAd-vectorhastobeelucidatedindetailandindesirableinteractionsshouldbeavoidedoreliminatedtoenhancetransductiontodesiredtargetcells.Tothisend,attemptstoreducethehepatotropism ofAd5vectorshavebeen

basedon theassumption thatCAR-basedinteractionsarerequiredforliveruptakein vivo.Complexation with cationicliposomehasbeen attempted toinhibithepatocyteand/orliverKupffercelluptakeusingAd5vectors.Recentstudiesfocused on Adenoviralvectorbiodistribution havedemonstrated thatKupffercellandhepatocyteuptakeinvivoarelargelyCAR-independent(26,31).In thisregard,threedifferentcationicliposomescomplexed to adenoviral

vectors were systemically administered in B16BL6 tumor-bearing C57BL/6mice.The mice administered with adenoviral vectors alone exhibited arelativelyhigherluciferaseexpressionintheliverandotherorgansaswell.Aspreviously reported,thismay beresulted from Kupffercelland hepatocyteuptake in the liver.Meanwhile,complexation with DMKE-based cationicliposomes yielded remarkably differentbiodistribution patterns.The hepatictransduction ofadenoviralvectors was greatly reduced by addition oftheDMKE cationicliposomes.Unfortunately,thereductionofhepatictransductionwas notcompensated in any otherorgans including tumors.In fact,thecomplexation with the DMKE liposomes showed 200-fold lowertransgeneexpressionintheliverwhileonlyslightlyhigherexpressionintumors.ItisconceivablethatthedifferentsurfacenatureofAd-liposomescomplexesmayprovide a different biodistribution pattern. The adenoviral particles are

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electronegatively charged while the Ad-liposomes complexed areelectropositivelycharged.Therefore,theiropsonizationinbloodwillbedifferentfrom eachother,whichwillresultindifferentpatternsofcirculationinthebody.At least,the in vivo data in this study clearly also show thatcomplexation with the DMKE-based cationic liposomes can reduce thehepatotropism.A numberofstudieshavereportedthatAd-liposomecomplexescanincrease

transduction efficiency in celllinesthatareresistanttoadenoviralinfection(32-34).To my knowledge,this is the first study to look at in vivotransduction by Ad-liposome complexes. The DMKE-based liposomescomplexed to Ad-vectors were able to reduce in vivo hepatotropism ofAd-vectors. In addition, complexation with the cationic liposomes alsodisplayed CAR-independenttransduction by Ad-vectorsin thetestedcancercells resistantto adenoviralinfection.This implied thatthe DMKE-basedcationicliposomalmembranescoating adenoviralparticlesmay providemoreeffectiveinteractionswithhostcells.However,atthismomenttheliposomalformulationpreparedforthisstudyisnotappropriateforsystemicdeliveryofAd-vectorstoany intendedorgansortissues,suchastumors.In ordertodevelopanAd-liposomecomplex system fortarget-specifictransduction,theliposomalsystem hastobefurtheroptimizedin termsoflipidcomposition,surfacecharge,andparticlesize.Conjugationoftargetingligands,cell-specificantibodiesandpeptides,willbealsohelpfultodeliveryadenoviralparticlestotargetcells.

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ⅤⅤⅤ...RRREEEFFFEEERRREEENNNCCCEEESSS1.NakayamaM,BothGW,BanizsB,TsurutaY,YamamotoS,KawakamiY,Douglas JT,TaniK,CurielDT,Glasgow JN (2006) An adenovirusserotype5vectorwithfibersderivedfrom ovineatadenovirusdemonstratesCAR-independenttropism anduniquebiodistributioninmice.Virology350(1),103-115.

2.BergelsonJM,Cunningham JA,DroguettG,Kurt-JonesEA,KrithivasA,Hong JS,Horwitz MS,CrowellRL,Finber,RW (1997)Isolation ofacommon receptorforCoxsackie B viruses and adenoviruses 2 and 5.Science275(5304),1320-1323.

3.ButtgereitP,Weineck S,Ropke G,Marten A,Brand K,Heinicke T,CaselmannWH,HuhnD,Schmidt-WolfIGH (2000)Efficientgenetransferinto lymphoma cells using adenoviralvectorscombined with lipofection.CancerGeneTherapy7(8),1145-1155.

4.MizuguchiH,HayakawaT (2004)Targetedadenovirusvectors(Review).Humangenetherapy15(11),1034-1044.

5.EinfeldDA,SchroederR Roelvink,PW,LizonovaA,KingCR,KovesdiI,Wickham TJ(2001).Reducing thenativetropism ofadenovirusvectorsrequiresremovalofboth CAR and integrin interaction.TheJournalofVirology75(23),11284-11291.

6.LeissnerP,LegrandB,SchlesingerY,HadjiDA,vanRaaijM,CusackS,PaviraniA,MehtaliM (2001) Influenceofadenoviralfibermutationsonviralencapsidation,infectivity and in vivotropism.Genetherapy 8(1),49-57.

7.Alemany R,CurielDT (2001) CAR-binding ablation does notchangebiodistribution and toxicity ofadenoviralvectors.Gene therapy 8 (17),1347-1353.

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8.Wang Y,Hu JK,Krol1 A,LiYP,Li,CY,Yuan F (2003)Systemicdissemination of viralvectors during intratumoralinjection.Molecularcancertherapy2(11),1233-42.

9.Wickham TJ(2000).Targetingadenovirus.GeneTherapy7(2),110-114.10.BykT,HaddadaH,VainchenkerW,LouacheF (1998)Lipofectamineandrelated cationic lipids strongly improve adenoviralinfection efficiency ofprimitivehumanhematopoieticcells.Humangenetherapy9(17),2493-502.

11.FasbenderAI,ZabnerJ,ChillonM,MoningerTO,PugaAP,DavidsonBL,WelshMJ(1997)Complexesofadenoviruswithpolycationicpolymersandcationiclipidsincreasetheefficiencyofgenetransferinvitroandinvivo.JournalofBiologicalChemistry272(10),6479-6489.

12.LeeSG,Yoon너,Kim CD,Kim K,Lim DS,Yeom YI,SungMW,HeoDS, Kim NK (2000) Enhancement of adenoviral transduction withpolycationicliposomesinvivo.Cancergenetherapy 7(10),1329-35.

13.ToyodaK,NakaneH,HeistadDD (2001)Cationicpolymerandlipidsaugmentadenvirus-mediated gene transfer to cerebralarteries in vivo.JournalofCerebralBloodFlow & Metabolism 21(9),1125-31.

14.YotndaP,ChenDH,ChiuW,PiedraPA,DavisA,TempletonNS,BrennerMK (2002)Bilamellarcationicliposomesprotectadenovirusfrom preexistinghumoralimmuneresponses.MolecularTherapy5(3),233-241.

15.Mok KW,Lam AM,CullisPR (1999)Stabilized plasmid-lipid particles:factors influencing plasmid entrapment and transfection properties.BiochimicaetBiophysicaActa(BBA)/Biomembranes1419(2),137-150.

16.LeeEM,HongSH,LeeYJ,KangYH,ChoiKC,ChoiSH,Kim IH,Lim SJ(2004) Liposome-complexed adenoviral gene transfer in cancer cellsexpressingvariouslevelsofcoxsackievirusandadenovirusreceptor.JournalofCancerResearchandClinicalOncology 130(3),169-177.

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17.Miller,C.R.,Buchsbaum,D.J.,Reynolds,P.N.,Douglas,J.T.,Gillespie,G.Y.,MayoMS,RabenD,LurielDT (1998)Differentialsusceptibilityofprimaryandextablishedhumangliomacellstoadenovirusinfection:targetingviatheepidermalgrowth factorreceptorachievesfiberreceptor-independentgenetransfer.CancerResearch58(24),5738-5748.

18.HemminkiA,Alvarez RD (2002)Adenoviruses in oncology :a viableoption?BioDrugs16(2),77-87.

19.BauerschmitzG.J,BarkerSD,HemminkiA (2002)Adenoviralgenetherapyfor cancer:from vectors to targeted and replication competentagents(Review).Int.JournalofOncology 21(6),1161-1174.

20.TaoN,GaoGP,ParrMJ,BaradetT,WilsonJM,Barsoum J,FawellSE(2001)Sequesteration ofadenoviralvectorby Kupffercells leads to anonlineardoseresponseoftransductioninliver.Moleculartherapy3(1),28-35.

21.Connelly S (1999)Adenoviralvectors for liver-directed gene therapy.CurrentOpinioninMolecularTherapeutics1(5),565-572.

22. Peeters MJ, Patijn GA, Lieber A, Meuse L, Kay MA (1996)Adenovirus-mediated hepatic gene transfer in mice: comparison ofintravascular and biliary administration.Human Gene Therapy 7 (14),1693-1699.

23.SulivanDF,DashS,DuH,HiramatsuN,AydinF,KollsJ,BlanchardJ,BaskinG,GerberMA (1997)Liver-directedgenetransferinnon-humanprimates.HumanGeneTherapy8(10),1195-1206.

24.WorgallS,WolffG,Falck-PedersenE,Crystal,RG (1997)Innateimmunemechanismsdominateelimination ofadenoviralvectorsfollowing in vivoadministration.HumanGeneTherapy.8(1),37-44.

25.Alemany,R.,Suzuki,K,Curiel,D.T.,(2000)Blood clearance rates ofadenovirus type 5 in mice.JournalofGeneralVirology 81 (Pt,11),

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2605-2609.26.ShayakhmetovDM,LiZY,NiS,LieberA (2004)Analysisofadenovirussequestrationintheliver,transductionofhepaticcells,andinnatetoxicityafterinjectionoffiber-modifiedvectors.TheJournalofVirology78(10),5368-5381.

27.FechnerH,Haack A,Wang H,Wang X,Eizema K,PauschingerM,SchoemakerRG,VeghelRV,HoutsmullerAB,SchultheissHP,LamersJMJ,Poller W (1999) Expression of coxsackie adenovirus receptor andalphav-integrin dose not correlate with adenovector targeting in vivoindicatinganatomicalvectorbarriers.GeneTherapy6(9),1520-1535.

28.ReynoldP,DmitrievI,CurielD (1999)InsertionofanRGD motifintotheHIloop ofadenovirus fiberprotein alters the distribution oftransgeneexpression ofthesystemically administered vector.GeneTherapy 6(7),1336-1339.

29.WoodM,PerrotteP,OnishiE,HarperME,DinneyC,PagliaroL,WilsonDR (1999)Biodistribution ofan adenoviralvectorcarrying theluciferasereporter gene following intravesicalor intravenous administration to amouse.CancerGeneTherapy6(4),367-372.

30.LieberA,HeCY,MeuseL,SchowalterD,KirllovaI,WintherB,KayMA(1997)TheroleofKupffercellactivation and viralgeneexpression inearlylivertoxicityafterinfusionofrecombinantadenovirusvectors.TheJournalofVirology71(11),8798-8807.

31.LiuQ,ZaissAK,ColarussoP,PatelK,HaljanG,Wickham TJ,MuruveDA (2003)Theroleofcapsid-endothelialinteractionintheinnateimmuneresponsetoadenovirusvectors.Hum GeneTherapy14(7),627-643.

32.ToyodaK,OoboshiH,Chu Y,FasbenderA,Davidson BL,Welsh MJ,HeistadDD(1998)Cationicpolymerandlipidsenhanceadenovirus-mediatedgenetransfertorabbitcarotidartery.Stroke29(10),2181-2188.

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33.RyukeY,MizunoM,NatsumeA,YoshidaJ(2000)Transductionefficiencyofadenoviralvectorsintohumangliomacellsincreasedbyassociationwithcationicliposomes.Neurologiamedico-chirurgica40(5),256-260.

34.FukuharaH,HayashiY,YamamotoN,FukuiT,NishikawaM,MitsudoK,Tohnai I, Ueda M, Mizuno M, Yoshida J (2003) Improvement oftransduction efficiency ofrecombinantadenovirusvectorconjugated withcationicliposomeforhumanoralsquamouscellcarcinomacelllines.OralOncology39(6),601-609.

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국국국문문문 요요요약약약

양양양이이이온온온성성성 리리리포포포솜솜솜이이이 아아아데데데노노노바바바이이이러러러스스스성성성 벡벡벡터터터의의의 세세세포포포내내내 및및및체체체내내내 유유유전전전자자자 전전전달달달에에에 미미미치치치는는는 영영영향향향

연세대학교 대학원임상병리학과

이 연경

아데노바이러스 벡터는 유전자 발현효율이 좋아 유전자 치료에 널리 사용되고 있다. 하지만 이러한 아데노바이러스 벡터는 세포감염을 위해서 CAR(coxsackieadenovirusreceptor)의 발현을 필요로 하며,특히 체내에서는 간,비장등의 장기에 비특이적으로 축적되는 단점을 가지고 있다.따라서 본 논문에서는이러한 단점을 극복하기 위해,다양한 조성의 양이온성 리포솜과 아데노바이러스벡터를 결합(complexation)시켜 세포내 그리고 체내 유전자전달(transduction)양상을 확인하고자 하였다.양이온성 리포솜은 DMKE(O,O’-dimyristyl-glutamyl-lisine), DMKE/Chol(cholesterol) (60:40 molar ratio), DMKE/Chol/Gal-Cer(galactosylceramide)(60:35:5,몰비율)세 가지로 제조되었다.일반적으로 CAR를발현하지 않는 B16BL6세포와 MCF-7세포에서는 양이온성 리포솜이 아데노바이

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러스 벡터의 유전자전달활성을 향상시켰다.반면에 CAR를 발현하여 아데노바이러스에 의해 유전자전달이 비교적 잘되는 HeLa,HepG2,SNU739세포에서는 다양한 영향을 주었다.양이온성 리포솜이 HeLa세포에서의 유전자전달에 거의 영향을 주지 않았거나 HepG2 세포에서는 유전자전달효율을 감소시켰던 반면에SNU793 세포에서는 유전자전달효율을 향상시켰다. B16BL6 세포가 이식된C57BL/6동물 모델에 아데노바이러스 벡터만을 투여한 결과 간조직에서 가장 높은 유전자발현을 볼 수 있었다.반면에 양이온성 리포솜을 결합한 아데노바이러스복합체를 투여한 경우에는 간에서의 유전자발현이 최고 200배의 이상의 감소하였다.이러한 간에서 유전자발현 감소에도 불구하고 암조직을 포함한 여타 조직에서의 유전자발현은 증가되지 않았다.DMKE/Chol,DMKE/Chol/Gal-Cer리포솜 역시 간에서의 유전자발현을 억제하였을 뿐 아니라 다른 장기에서 유전자발현 또한감소하였다.또한 HeLa세포가 이식된 누드마우스에서도 아데노바이러스는 간과비장에서 높은 유전자 발현을 유도하였다.같이 투여된 DMKE/Chol/Gal-Cer리포솜 역시 간과 비장에서 유전자 발현을 억제하였다.본 연구를 통해 아데노바이러스 벡터에 양이온성 리포솜을 결합시킴으로써 대상세포의 CAR발현에 상관없이효과적인 유전자발현을 유도할 수 있으며,생체 내에서는 간과 비장으로의 축적을감소시킬 수 있었다.

핵심어: 아데노바이러스성 벡터, 유전자 치료, 양이온성 리포솜, coxsackieadenovirus수용체(CAR),MCPS(mononuclearcellphagocyticsystem)

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감감감사사사의의의 글글글

학부 졸업 후 공백기를 거쳐 다시 대학원이라는 곳에 발 들여 놓은 것이 엊그제 같은데 벌써 2년이라는 시간이 훌쩍 지나가 버렸습니다.2년 동안 많은 것을배우고 느끼게 해주었던 시간이었습니다.바쁘신 와중에도 논문 시작부터 교정까지 지도해주시고 신경 써주신 박용석 교수님께 깊이 감사드립니다.관심과 애정을 가지시고 지도해주신 양용석 교수님,오옥두 교수님,김종배 교수님,김태우 교수님,이혜영 교수님께 진심으로 감사드립니다.또한 실험실 전반에 걸쳐 관심을 가지고 지도해주신 임병혁 선생님께 감사드립니다.실험에 필요한 기자재 및 조언을 아낌없이 주신 원자력 병원의 최태현선배님과 밤늦은 시간까지 같이 실험에 도움을 주신 삼성 의료원 문철 선배님,외국에서 바쁘신 와중에도 친절히 질문에 답해주신 근식 오빠,2년간 대학원 생활을하면서 여러모로 가르쳐주고 조언 해주신 진숙언니,인수 오빠,규상 오빠,방혜은선생님,현철 오빠,은아 언니,은주 선배,연임 선배,관훈 선배,처음 입학해서 어리버리한 후배 잘 챙겨주고 논문 쓸 때 도움 많이 준 인호선배,다른 할 일 많은데도 불구하고 부탁할 때마다 거절 없이 도와준 정석,후배지만 힘들 때마다 내옆에서 친구처럼 같이 고민해주고 귀 기울여 주던 경혜,힘든 대학원 생활에서 언제나 웃음과 도움을 주었던 착한 후배들 정례,혜은,실험하러 갈 때 마다 자신들의 일인 것처럼 3인 1조가 되어 휴일도 잊어버린 채 실험에 도움을 준 수희,종찬,수다친구 도완,착한 형엽 오빠,상정,M&D 선생님들,실험실에서 뭐든지 열심인화연,모두에게 감사드립니다.동기 없이 시작한 대학원 생활이었지만 외롭지 않고 힘들지만 이겨낼 수 있었던것은 모두 선배님 후배님들의 관심과 위로 덕분이었습니다.무엇보다도 항상 기도로 저를 보살펴 주시며 든든한 후원자가 되어주신 저의 부모님과 사랑하는 동생수진 에게 깊은 감사의 마음을 전하고 싶습니다.

CONTENTSABSTRACTⅠ.INTRODUCTIONⅡ.MMAATERIALSAND MMEETHODS1.Materials2.Celllinesandcellculture3.Preparationofcationicliposomes4.RT-PCRanalysisforCARexpressiononthesurfaceofcancercells5.Invitroadenoviraltransduction6.AnalysisofinvitroGFPexpression7.Cytotoxicityassayofcationicliposomes8.Invivogenetransductionandanalysisoftransgeneexpression

Ⅲ.RESULTS1.CARexpressiononthesurfaceofcancercells2.Invitrotransductionmediatedbyadenoviralvectorandadenovirus-liposomecomplexes3.CytotoxicityofcationicliposomescomplexedtoAd-vectors4.InvitrotransfectionbycationicliposomesandtransductionbyAd-liposomecomplexes5.InvitrogenetransductionmediatedbyAd-liposomecomplexesinvariedcelllines6.InvivotransductionbyAd-vectorsandAd-liposomecomplexes

Ⅳ.DISCUSSIONⅤ.REFERENCES국문 요약