Tumor-targeted nanoparticle delivery of HuR siRNA ...

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

7XPRUWDUJHWHG QDQRSDUWLFOH GHOLYHU\ RI +X5 VL51$ LQKLELWV OXQJ WXPRU JURZWK in vitro DQG in vivo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in vitroDQGin vivo>@ )RU HIIHFWLYH in vivo OXQJ FDQFHU WKHUDS\ XVLQJ +X5 VL51$ ZH FUHDWHG D WDUJHWHG GHOLYHU\ V\VWHP E\ PRGLI\LQJ WKH '27$3&KRO QDQRSDUWLFOH SODWIRUP ZLWK WUDQVIHUULQ DV D WDUJHWLQJOLJDQG'63(3(*7I 7KHWDUJHWLQJPRLHW\7IZDVFKRVHQEDVHGRQWKHH[SUHVVLRQ OHYHOV RI LWV UHFHSWRUV 7I5 RU &'  LQ VROLG DQG PHWDVWDWLF OXQJ WXPRU PRGHOV ,QLWLDOO\ ZH WHVWHGWKHHIILFLHQF\RI+X57I13versus+X513QRQWDUJHWHG in vitro%DVHGRQWKHUHVXOWV REWDLQHGZHK\SRWKHVL]HGWKDW7I13EDVHGWDUJHWHGGHOLYHU\RI+X5VL51$ZRXOGUHGXFHWKH WXPRU EXUGHQ LQ PRXVH PRGHOV RI OXQJ FDQFHU :H VWXGLHG 7I13 V ELRGLVWULEXWLRQ LQ PRXVH PRGHOVDQGUHO\LQJRQWKLVGDWDZHHYDOXDWHGWKHWDUJHWVSHFLILFLW\DQGHIILFDF\RI+X57I13 versus &7I13 FRQWURO VL51$  LQ VROLG WXPRU PRGHOV )LQDOO\ ZH XVHG in vivo OLYH LPDJLQJ WXPRU QRGXOH FRXQWV DQG LPPXQRKLVWRFKHPLVWU\ RI VSHFLILF PROHFXODU PDUNHUV WR LQYHVWLJDWH ϱ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

WXPRU JURZWK LQKLELWLRQ DQG WKH DQWLPHWDVWDWLF DFWLYLW\ RI +X57I13 LQ D PRXVH PRGHO RI PHWDVWDWLF$OXFOXQJFDQFHU   0DWHULDOVDQG0HWKRGV &KHPLFDOV GLROHR\OWULPHWK\ODPPRQLXPSURSDQH FKORULGH '27$3  FKROHVWHURO DQG GLVWHDUR\OsnJO\FHURSKRVSKRHWKDQRODPLQH1>SRO\HWK\OHQH

JO\FRO @

'63(

3(*  ZHUH SXUFKDVHG IURP $YDQWL 3RODU /LSLGV $ODEDVWHU $/  7UDQVIHUULQ DQG GHVIHUULR[LPLQH ZHUH SXUFKDVHG IURP 6LJPD$OGULFK &KHPLFDOV 6W /RXLV 02  530, PHGLXP(0(0PHGLXPDQGIHWDOERYLQHVHUXP)%6 ZHUHSXUFKDVHGIURP*,%&2%5//LIH 7HFKQRORJLHV1HZ@ 7KH HOHFWURSKRUHVLV JHO VWDLQHG ZLWK HWKLGLXP EURPLGH 6,*0$ 6W /RXLV 02  ZDV YLVXDOL]HG DQG LPDJHG XVLQJ D 6\QJHQHJHOGRFXPHQWDWLRQV\VWHP SiRNA release profile7I13ORDGHGZLWKVL*/2ZHUHVXVSHQGHGLQWLVVXHFXOWXUHPHGLXPS+   DFHWDWH EXIIHU $%6 S+   RU WLVVXH FXOWXUH PHGLXP ZLWK  IHWDO ERYLQH VHUXP DQG ZHUHLQFXEDWHGDWƒ&ZLWKJHQWOHVKDNLQJDWUSP7KHUHOHDVHRIVL51$ZDVPRQLWRUHGDW VHYHUDOWLPHLQWHUYDOVDVGHVFULEHGSUHYLRXVO\>@  2SWLPL]DWLRQ RI 7I3(*'63( RQ '27$3&KRO :H HYDOXDWHG WKH FHOOXODU XSWDNH RI 7I13LQ$FHOOVZLWKUHVSHFWWRPRGLILFDWLRQUDWLR$[ZHOO FHOOVVHHGHGLQDVL[ ϴ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

ZHOOSODWHZHUHWUDQVIHFWHGZLWKDVL*/27I13FRPSOH[FRQWDLQLQJRU PRO RI 7I3(*'63( WR WKH WRWDO OLSLG LQ VHUXPIUHH PHGLXP IRU VL[ KRXUV $IWHU WUDQVIHFWLRQ FHOOV ZHUH UHSOHQLVKHG ZLWK 530, FRQWDLQLQJ  )%6 DQG LQFXEDWLRQ ZDV FRQWLQXHG &HOOV ZHUH KDUYHVWHG DW  K DIWHU WUDQVIHFWLRQ DQG IOXRUHVFHQFH H[SUHVVLRQ ZDV GHWHUPLQHGXVLQJDQ(QYLVLRQŠSODWHUHDGHU3HUNLQ(OPHU6DQWD&ODUD&$ &HOOVWKDWZHUHQRW WUDQVIHFWHGRUWUDQVIHFWHGZLWKRXWIUHHOLJDQGVHUYHGDVFRQWUROV 5HFHSWRUPHGLDWHG HQGRF\WRVLV $ [ FHOOVZHOO  ZHUH VHHGHG LQ D VL[ZHOO SODWH DQG ZHUHWUHDWHGZLWKVL*/27I138QWUHDWHGFHOOVVHUYHGDVFRQWUROV$IWHU13WUHDWPHQWWKHSODWHV ZLWKFHOOVZHUHLQFXEDWHGLQHLWKHUƒ&RUƒ&>@7KHFHOOVZHUHKDUYHVWHGDIWHUKDQGK RILQFXEDWLRQDQGWKHIOXRUHVFHQFHLQWHQVLW\GHWHUPLQHGXVLQJDQ(QYLVLRQŠSODWHUHDGHU  6SHFLILFLW\VWXGLHV Receptor stimulation studies$FHOOV[ZHOO ZHUHVHHGHGLQVL[ZHOOSODWHV$IWHUK RILQFXEDWLRQWKHPHGLXPZDVUHSODFHGZLWKPORIVHUXPIUHHPHGLXPZLWKRUZLWKRXW —0RIGHVIHUULR[DPLQH')2  $IWHUKRILQFXEDWLRQWKH PHGLD ZDV UHSODFHG ZLWK VL*/2 7I13FRQWDLQLQJPHGLD$IWHUKWKLVVL*/27I13FRQWDLQLQJPHGLDZDVUHSODFHGZLWKIUHVK  VHUXP FRQWDLQLQJ PHGLD DQG WKH FHOOV ZHUH LQFXEDWHG RYHUQLJKW 1H[W GD\ WKH FHOOV ZHUH KDUYHVWHGDQGWKHIOXRUHVFHQFHLQWHQVLW\RIVL*/2ZDVPHDVXUHGXVLQJ(QYLVLRQŠSODWHUHDGHU Receptor blocking studies $ FHOOV [ FHOOVZHOO  VHHGHG LQ VL[ZHOO SODWHV ZHUH SUHWUHDWHGZLWKRUZLWKRXWKXPDQWUDQVIHUULQ+7IPJ DQGZHUHLQFXEDWHGDWƒ&ZLWK &2 IRU  K 7KH FHOOV ZHUH VXEVHTXHQWO\ WUHDWHG ZLWK VL*/27I13 LQ VHUXPIUHH PHGLXP ,QFXEDWLRQ FRQWLQXHG IRU DQ DGGLWLRQDO   DQG  K $W WKH HQG RI WKHLQFXEDWLRQ SHULRGWKH FHOOVZHUHVXEMHFWHGWRIOXRUHVFHQFHLQWHQVLW\PHDVXUHPHQW ϵ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

,Q DQRWKHU VHW RI H[SHULPHQWV $ FHOOV [ FHOOV ZHOO  VHHGHG LQ VL[ZHOO SODWHV

ZHUHSUHWUHDWHGZLWKRUZLWKRXW—ORIKXPDQWUDQVIHUULQDQWLERG\DQGZHUHLQFXEDWHGDWƒ& ZLWK  &2 IRU  K 7KH FHOOV ZHUH VXEVHTXHQWO\ WUHDWHG ZLWK VL*/27I13 LQ VHUXPIUHH PHGLXP,QFXEDWLRQFRQWLQXHGIRUDQDGGLWLRQDOKDQGK$WWKHHQGRIWKHLQFXEDWLRQSHULRG WKHFHOOVZHUHVXEMHFWHGWRIOXRUHVFHQFHLQWHQVLW\PHDVXUHPHQW  &HOOXODU XSWDNH VWXGLHV &HOOXODU LQWHUQDOL]DWLRQ RI 7I13 ORDGHG ZLWK IOXRUHVFHQWO\ ODEHOOHG VL51$ZDVPHDVXUHGTXDQWLWDWLYHO\ZLWKDQ(QYLVLRQŠSODWHUHDGHU$+&&DQG05& FHOOV ZHUH VHSDUDWHO\ VHHGHG LQ VL[ZHOO SODWHV DW [ FHOOV ZHOO 7KH QH[W GD\ FHOOV ZHUH WUDQVIHFWHGZLWKHLWKHUVL*/27I13RUVL*/213LQVHUXPIUHHPHGLXP&HOOVZHUHKDUYHVWHG DIWHUKKDQGKRILQFXEDWLRQDQGZHUHDQDO\]HGIRUWKHPHDQIOXRUHVFHQFHLQWHQVLW\  &HOOYLDELOLW\DVVD\$+&&DQG05&FHOOVZHUHVHHGHGLQDVL[ZHOOSODWHDW[ LQ  PO RI 530, DQG (0(0 PHGLD FRQWDLQLQJ  )%6 $IWHU  K RI LQFXEDWLRQ LQ D KXPLGLILHGDWPRVSKHUHRI&2DWƒ&WKHPHGLXPZDVUHSODFHGZLWKPORIVHUXPIUHH PHGLXPDQGZDVWUHDWHGZLWK7I13ORDGHGZLWKFRQWUROVL51$&7I13 RU+X5VL51$+X5 7I13  7KHILQDOFRQFHQWUDWLRQRIVL51$XVHGLQWKHVHH[SHULPHQWVZDVQ0$IWHUKRI LQFXEDWLRQWKHPHGLXPZDVUHSODFHGZLWK530,DQG(0(0PHGLXPFRQWDLQLQJVHUXPDQG ZDVLQFXEDWHGIRUDQDGGLWLRQDOKDQGKDWƒ&&HOOVZHUHKDUYHVWHGDQGFHOOYLDELOLW\ ZDVGHWHUPLQHGZLWKWU\SDQEOXHH[FOXVLRQDVVD\>@ 

ϭϬ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

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—JRIWRWDO51$XVLQJ4XDQWVFULSWF'1$V\QWKHVLVNLW%LR-5DG, 5LFKPRQG&$ 7KH F'1$ ZDV VXEVHTXHQWO\ XVHG WR SHUIRUP T573&5 %LR5DG &); 7RXFK 5HDO7LPH 3&5 'HWHFWLRQ 6\VWHP ZLWK6@8QWUHDWHGFHOOVVHUYHGDVFRQWUROV  0LJUDWLRQ DQG LQYDVLRQ DVVD\ 7KH FHOO PLJUDWLRQ DQG LQYDVLRQ DVVD\ ZDV FDUULHG RXW DV SUHYLRXVO\ GHVFULEHG >  @ %ULHIO\ &7I13 DQG +X57I13WUHDWHG $ FHOO ZHUH ϭϭ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

VHHGHGLQWKHXSSHUFKDPEHUVRIWKHPLJUDWLRQDQGLQYDVLRQLQVHUWV&HOOVUHFHLYLQJQRWUHDWPHQW VHUYHG DV FRQWUROV 7KH ORZHU FKDPEHU ZDV ILOOHG ZLWK  )%6FRQWDLQLQJ FXOWXUH PHGLXP $IWHUKDQGKRILQFXEDWLRQWKHQXPEHURIPLJUDWHGDQGLQYDGHGFHOOVZHUHFRXQWHGDQG WKHUHVXOWVUHSUHVHQWHGDVSUHYLRXVO\GHVFULEHG>@ $QQH[LQ9DVVD\$FHOOV[ VHHGHGLQDVL[ZHOOSODWHZHUHWUHDWHGZLWK&7I13DQG +X57I13DQGVWDLQHGZLWKDQQH[LQ9FRQMXJDWHGWR$OH[D)OXRUG\HDQGSURSLGLXPLRGLGH 3, XVLQJ'HDGFHOODSRSWRVLVNLW/LIH7HFKQRORJLHV(XJHQH25 DFFRUGLQJWRPDQXIDFWXUHU¶V SURWRFRO %ULHIO\ WKH FHOOV ZHUH KDUYHVWHG DW  K DIWHU WUHDWPHQW DQG VXVSHQGHG LQ DQQH[LQ9 ELQGLQJ EXIIHUDWDFRQFHQWUDWLRQRIFHOOVPO —ORIFHOOVXVSHQVLRQZDVLQFXEDWHG ZLWK  —O RI DQQH[LQ9 DQG —O RI  —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n vivoVWXGLHV 6XEFXWDQHRXVWXPRU[HQRJUDIWDQGH[SHULPHQWDOOXQJPHWDVWDVLVPRGHOV)RXUWRVL[ZHHN ROG IHPDOH QXGH PLFH 1X1X  ZHUH SXUFKDVHG IURP &KDUOHV 5LYHU /DERUDWRULHV :LOPLQJWRQ 0$ 7KHVWXGLHVZHUHFRQGXFWHGZLWKWKHDSSURYDORIDQGLQDFFRUGDQFHZLWKJXLGHOLQHVRIWKH ,QVWLWXWLRQDO $QLPDO &DUH DQG 8VH &RPPLWWHH DW WKH 8QLYHUVLW\ RI 2NODKRPD +HDOWK 6FLHQFHV &HQWHU2NODKRPD&LW\2.)RUHVWDEOLVKLQJVXEFXWDQHRXVVF WXPRUVPLFHZHUHDQHVWKHWL]HG ϭϮ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

ZLWKLVRIOXUDQHDQGLQMHFWHGZLWK $ DQG +&& WXPRU FHOOV[FHOOV LQ —ORI 3%6  VXEFXWDQHRXVO\ LQ WKH ORZHU ULJKW IODQN RI PLFH )RU VWXGLHV LQYROYLQJ ELROXPLQHVFHQFH LPDJLQJ$VWDEO\H[SUHVVLQJOXFLIHUDVHOXF$OXF ZDVXVHG:KHQWKHWXPRUVUHDFKHG DVL]HRIDERXWPPWKHPLFHZHUHUDQGRPL]HGDQGXVHGIRUELRGLVWULEXWLRQDQGHIILFDF\ VWXGLHVDVGHVFULEHGEHORZ )RU HVWDEOLVKLQJ H[SHULPHQWDO OXQJ PHWDVWDVLV $OXF FHOOV [ FHOOV LQ  —O RI 3%6 ZHUHLQMHFWHGLQWUDYHQRXVO\LY YLDWDLOYHLQ7XPRUIRUPDWLRQLQWKHOXQJVZDVGHWHFWHG E\ LQWUDSHULWRQHDOO\ LS  LQMHFWLQJ 'OXFLIHULQ '/XF  PJNJ  DQG PHDVXULQJ ELROXPLQHVFHQFH XVLQJ WKH ,9,6Š 6SHFWUXP ,PDJLQJ 6\VWHP 3HUNLQ (OPHU  :KHQ WKH WXPRU ELROXPLQHVFHQFHVLJQDOVUHDFKHGDSSUR[LPDWHO\DYHUDJHUDGLDQFHRISVFPðVUPLFHZHUH UDQGRPO\DVVLJQHGWRWUHDWPHQWJURXSVDQGHIILFDF\VWXGLHVFRQGXFWHG  %LRGLVWULEXWLRQVWXG\)RUGHWHUPLQLQJWKH7I13ELRGLVWULEXWLRQin vivo,LQGRF\DQLQHJUHHQDQ DPSKLSKLOLFFDUERF\DQLQHJUHHQG\H6LJPD$OGULFK0LVVRXUL WKDWIOXRUHVFHVDWWKHQHDULQIUD UHG 1,5  UHJLRQ ZDV HQFDSVXODWHG LQ 7I13V 7KHVH 13V ZHUH SUHSDUHG IROORZLQJ WKH VDPH PHWKRG IRU HQFDSVXODWLQJ WKH VL51$ DV GHVFULEHG DERYH IRU in vitro VWXGLHV ZLWK PLQRU PRGLILFDWLRQV %ULHIO\ DQ HTXDO YROXPH RI '27$3&KRO  P0  VWRFN VROXWLRQ DQG ,&* VROXWLRQPJPOLQZDWHU ZDVPL[HGYRUWH[HGDQGLQFXEDWHGDWURRPWHPSHUDWXUHIRUPLQ '63(3(*7I OLJDQGV ZHUH LQVHUWHG LQWR SUHIRUPHG OLSRVRPHV XVLQJ WKH SRVW LQVHUWLRQ WHFKQLTXH7KHVROXWLRQVZHUHWKHQGLDO\]HGDJDLQVWZDWHURYHUQLJKWXVLQJDN'D0:FXWRII PHPEUDQHDQGZHUHFROOHFWHGDQGODEHOOHGDV,&*7I13 ,&*7I13  —J RI ,&*  ZDV DGPLQLVWHUHG LY YLD WDLO YHLQ WR PLFH EHDULQJ VXEFXWDQHRXV$DQG+&&WXPRUVRUWRQRQWXPRUEHDULQJQDwYHPLFH)UHH,&*LQMHFWHG ϭϯ

 Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

6XEFXWDQHRXVWXPRUEHDULQJPLFHZHUHXVHGDVFRQWUROV0LFHZHUHLPDJHG([QP(P QP DWPLQPLQKDQGKLQWHUYDOVSRVWLQMHFWLRQXVLQJ,9,6Š6SHFWUXP,PDJLQJ 6\VWHP 6LPLODUO\ IRU GHWHUPLQLQJ VL51$ ELRGLVWULEXWLRQ in vivo IOXRUHVFHQWO\ ODEHOHG '@ +X5 DQG .L VWDLQHG WXPRU WLVVXHV ZHUH DQDO\]HG XVLQJ D SRVLWLYH QXFOHDU FRXQW DOJRULWKP ZLWK WKH $SHULR ,PDJH6FRSH YLHZHU7KHQXPEHURISRVLWLYHQXFOHLIURPUHJLRQVRILQWHUHVW52,V ZDVGHWHUPLQHGIRUHDFK DQLPDO IURP &7I13 DQG +X57I13 WUHDWHG JURXSV )RU WKH &' PDUNHU VWDLQLQJ ZH GHWHUPLQHG WKH QXPEHU RI PLFURYHVVHOV SHU PP RI WKH WLVVXH XVLQJ WKH $SHULR PLFURYHVVHO DQDO\VLVDOJRULWKP7KHUHVXOWVREWDLQHGIRUHDFKRIWKHPDUNHULQWKHWZRWUHDWPHQWJURXSVZHUH VXEMHFWHGWRVWDWLVWLFDODQDO\VLV ϭϳ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

 781(/$VVD\781(/$VVD\ZDVSHUIRUPHGXVLQJLQVLWXFHOOGHDWKGHWHFWLRQNLWIURP5RFKH 'LDJQRVWLFV ,QGLDQDSROLV ,1  DFFRUGLQJ WR WKH PDQXIDFWXUHU¶V SURWRFRO %ULHIO\ WXPRU WLVVXH VHFWLRQV ZHUH LQFXEDWHG ZLWK SURWHLQDVH . IRU  PLQ EORFNHG DQG LQFXEDWHG ZLWK 781(/ UHDFWLRQ PL[WXUH IRU  PLQ DW  ƒ& $SRSWRWLF QXFOHL ZHUH ODEHOHG E\ WKH DGGLWLRQ RI DQWL IOXRUHVFHLQ DONDOLQH SKRVSKDWDVH FRQMXJDWH 6WDLQHG WXPRU VHFWLRQV ZHUH DQDO\]HG XVLQJ WKH $SHULR6FDQVFRSH,PDJHDQGWKHQXPEHURI781(/SRVLWLYHQXFOHLFRXQWHGXVLQJWKHSRVLWLYH QXFOHLFRXQWDOJRULWKPZLWKWKH$SHULR,PDJH6FRSHYLHZHU  6WDWLVWLFDO$QDO\VLV 7KH 6$6  VRIWZDUH ZDV XVHG IRU VWDWLVWLFDO DQDO\VHV $OO GDWD ZHUH SURYLGHG DV PHDQ “ VWDQGDUGGHYLDWLRQ6' 8QLYDULDWHVWDWLVWLFDOVLJQLILFDQFHZDVGHWHUPLQHGE\RQHZD\DQDO\VLV RIYDULDQFH$129$ ZLWK7XNH\¶VDGMXVWPHQWIRUSDLUZLVHFRPSDULVRQV'LIIHUHQFHVEHWZHHQ JURXSVZHUHREWDLQHGXVLQJDOLQHDUPL[HGHIIHFWVPRGHOZLWK7XNH\¶VDGMXVWPHQW$PYDOXHRI OHVVWKDQZDVFRQVLGHUHGVWDWLVWLFDOO\VLJQLILFDQW  5HVXOWV 3K\VLFRFKHPLFDOFKDUDFWHUL]DWLRQRI7I13 &DWLRQLF OLSLGEDVHG QDQRSDUWLFOHV DUH WKH PRVW FRPPRQO\ XVHG FDUULHUV IRU JHQH WKHUDSHXWLFVGXHWRWKHLUHIIHFWLYHJHQHWUDQVIHFWLRQDELOLW\in vitroDQGin vivo+HQFHZHFKRVH DFDWLRQLFOLSRVRPHSODWIRUP'27$3&KROZKLFKGHPRQVWUDWHGJHQHGHOLYHU\SRWHQWLDOLQRXU SUHYLRXVVWXGLHV>@LQIRUPXODWLQJ7I5WDUJHWHGQDQRSDUWLFOHV7I13 +X5VL51$ZDV DOORZHG WR LQWHUDFW HOHFWURVWDWLFDOO\ ZLWK FDWLRQLF '27$3&KRO OLSRVRPHV WR IRUP +X513 ϭϴ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

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p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“QPHPSW\13 WR “QP+X57I13 GXHWRVL51$ORDGLQJDQGVXUIDFHPRGLILFDWLRQZLWK7IOLJDQG>7DEOH@ 7KHVL51$ZDVZHOOFRQGHQVHGE\7I13UHVXOWLQJLQPRELOLW\UHWDUGDWLRQDVVKRZQLQWKHJHO HOHFWURSKRUHWRJUDP>6XSSOHPHQWDU\)LJ6E @7KHVL51$HQFDSVXODWLRQHIILFLHQF\H[SUHVVHG DVSHUFHQW(( ZDVFDOFXODWHGXVLQJWKHIROORZLQJHTXDWLRQ

ϭϵ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

 ((  >VL51$ DGGHG WR WKH OLSRVRPH IUHH XQHQWUDSSHG VL51$ VL51$ DGGHG WR WKH OLSRVRPH@ 7KHVL51$((ZDVIRXQGWREH“IRU+X57I13 7KH in vitro UHOHDVH SURILOHV RI siRNA-7I13 ZHUH LQYHVWLJDWHG LQ WKUHH GLIIHUHQW PHGLD FRQGLWLRQV VXFK DV DFHWDWH EXIIHU VROXWLRQ $%6 S+   WLVVXH FXOWXUHPHGLXP S+   DQG VHUXPFRQWDLQLQJFXOWXUHPHGLXPS+ DWƒ&RYHUDSHULRGRIK>6XSSOHPHQWDU\ )LJ 6F @ $ EXUVW VL51$ UHOHDVH SDWWHUQ ZDV REVHUYHG IRU WKH ILUVW  K a VL51$ UHOHDVHG LQFXOWXUHPHGLXPDQGVHUXPFRQWDLQLQJFXOWXUHPHGLXPS+ 7KHREVHUYHG VL51$UHOHDVHFKDUDFWHULVWLFVDUHW\SLFDORIDOLSLGQDQRSDUWLFOHGHOLYHU\V\VWHPDQGFRQFXUZLWK SUHYLRXVVWXGLHV>@+RZHYHUWKHVL51$UHOHDVHUDWHZDVKLJKHUZKHQVL51$7I13ZDV LQFXEDWHGLQFXOWXUHPHGLXPDORQHaVL51$UHOHDVHG FRPSDUHGWRVHUXPFRQWDLQLQJ PHGLXP a VL51$ UHOHDVHG  7KH VL51$ UHOHDVH ZDV QRWLFHDEO\ VORZHU DIWHU K XQWLO WKH PHDVXUHPHQW DW  K LQGLFDWLQJ D VXVWDLQHGUHOHDVHSDWWHUQ+RZHYHUDWORZS+ VL51$ UHOHDVH ZDV UDSLG UHDFKLQJ  LQ  K DQG  LQ  K 7KLV UHVXOW LV FRQVLVWHQW ZLWK RXU SUHYLRXVUHSRUWWKDWVKRZHQKDQFHGVL51$UHOHDVHUDWHDWORZS+ >@

In vitro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ϮϬ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

WUHDWHGJURXSLQGLFDWHVHQKDQFHGXSWDNHRIVL*/27I13FRPSDUHGZLWKFHOOVWKDWZHUHQRWSUH WUHDWHGZLWK')2 7R IXUWKHU XQGHUVWDQG WKH VSHFLILFLW\ RI 7I13 ZH FRQGXFWHG D VHSDUDWH VWXG\ LQ ZKLFK KXPDQ WUDQVIHUULQ +7I  DQG 7I13 ZHUH DOORZHG WR FRPSHWH DJDLQVW HDFK RWKHU >@ LQ LQWHUDFWLQJ ZLWK 7I5H[SUHVVLQJ $ FHOOV 7KH UHVXOWV VKRZHG WKDW +7I  —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ƒ& RU  ƒ& ZKLOH DGPLQLVWHULQJVL*/27I13WUHDWPHQW5HFHSWRUPHGLDWHGHQGRF\WRVLVLVNQRZQWREHDIIHFWHGE\ ORZWHPSHUDWXUH>@7KHUHVXOWVVKRZHGDPDUNHGGHFUHDVHHVSHFLDOO\DWKLQFHOOXSWDNHRI VL*/27I13DWƒ&FRPSDUHGZLWKƒ&VXJJHVWLQJWKDWWKHSUHGRPLQDQWPHFKDQLVPRIFHOO XSWDNHZDVviaUHFHSWRUPHGLDWHGHQGRF\WRVLV>6XSSOHPHQWDU\)LJ6G @ +DYLQJPDGHLWFOHDUWKDWWKH7I13LQWHUQDOL]DWLRQGHSHQGVRQ7I5H[SUHVVLRQOHYHOVLQ WKH WDUJHW FHOO ZH ZDQWHG WR YHULI\ ZKHWKHU 7I5 H[SUHVVLRQ OHYHOV FRQWULEXWH WR 7I13¶V VHOHFWLYLW\WRZDUGVOXQJFDQFHUFHOOVRYHUQRUPDOOXQJILEUREODVWFHOOV:HIRXQGWKDWERWK$ DQG +&& WXPRU FHOOV H[SUHVVHG VLJQLILFDQWO\ KLJKHU OHYHOV RI 7I5 WKDQ GLG QRUPDO 05& FHOOV)LJ%LQVHW $VH[SHFWHGWKHFHOOXSWDNHUHVSRQVHYDULHGDPRQJFDQFHUFHOOOLQHVDQG QRUPDOFHOOVDWKKDQGKWUHDWPHQWZLWKVL*/27I13XSWDNHFRUUHVSRQGLQJWRWKHLU7I5 H[SUHVVLRQ OHYHOV >)LJ %@ ,Q $ FHOOV WKH XSWDNH RI VL*/27I13 JUDGXDOO\ LQFUHDVHG DV Ϯϭ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

PHDVXUHG E\ IOXRUHVFHQFH LQWHQVLW\ DX  IURP   K  WR   K  DQG ODWHU WR Kp FRPSDUHGZLWKXQWUHDWHGFRQWURO7KRXJK+&&FHOOVDOVRVKRZHGD VLPLODUWUHQGLQFHOOXSWDNHRIVL*/27I13WKHIOXRUHVFHQFHLQWHQVLW\YDOXHVDWDOOWLPHSRLQWV ZHUHVLJQLILFDQWO\OHVVWKDQ$FHOOV,PSRUWDQWO\05&FHOOVVKRZHGQHJOLJLEOHFHOOXSWDNH RI VL*/27I13 DW DOO WLPH SRLQWV DQG ZDV VLJQLILFDQWO\ OHVV WKDQ WKH FDQFHU FHOO OLQHV VWXGLHG p   (IILFLHQF\RI7I13versus13LQVL+X5GHOLYHU\LQ7IRYHUH[SUHVVLQJOXQJFDQFHUFHOOV 6LQFH WKH WUDQVIHFWLRQ HIILFLHQF\ RI VL*/27I13 WDUJHWHG  RYHU XQWUHDWHG FRQWURO ZDV FRQILUPHGE\FHOOXSWDNHH[SHULPHQWVZHUHSODFHGVL*/2ZLWKVL51$DJDLQVW+X5WRWHVWLWLQ $FHOOV7KH+X5JHQHVLOHQFLQJHIIHFWLQGXFHGE\+X513QRQWDUJHWHG DQG+X57I13LQ $FHOOVZDVFRPSDUHGE\DQDO\]LQJWKH+X5DQGDVVRFLDWHGSURWHLQH[SUHVVLRQVDQGE\FHOO JURZWK LQKLELWLRQ $V VKRZQ LQ )LJ & JURZWK LQKLELWLRQ LQGXFHG E\ +X57I13 ZDV  FRPSDUHG ZLWK  IRU +X513 IRU D  K WUHDWPHQW SHULRG p   GHPRQVWUDWLQJ WKH VXSHULRUHIILFDF\RI+X57I13RYHU+X513 :HVWHUQEORWDQDO\VLVVKRZHGDGLIIHUHQFHLQ+X5H[SUHVVLRQEHWZHHQ+X513WUHDWHG +X5$FWLQ  DQG+X57I13WUHDWHG+X5$FWLQ p  JURXSV>)LJ'@7KLV ILQGLQJ VXJJHVWV WKDW WKH 7I13 IDFLOLWDWHG HQKDQFHG DQG PRUH HIILFLHQW NQRFNGRZQ RI +X5 FRPSDUHG ZLWK 13 7KH GDWD DOVR VKRZHG WKH HIIHFW RI +X5 NQRFNGRZQ LQ +X5UHJXODWHG SURWHLQVVXFKDV%&O&\FOLQ'DQG3+X5VWDELOL]HV%&OP51$DQGWKHUHE\WKH%&O IDPLO\RIDSRSWRWLFUHJXODWRU\SURWHLQVE\ELQGLQJWRLWV¶XQWUDQVODWHGUHJLRQ>@.QRFNGRZQ RI +X5 GHUHJXODWHV %&O UHGXFLQJ LWV H[SUHVVLRQ )LJ '  3UHYLRXVVWXGLHV KDYH VKRZQ WKDW GRZQUHJXODWLRQ RI +X5 UHVXOWHG LQ UHGXFHG H[SUHVVLRQ RI F\FOLQV ZKLFK DUH NH\ FHOO F\FOH ϮϮ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

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³+X5VL51$´DQGHQFDSVXODWHGLQ7I13 +X57I13 IRUFRQGXFWLQJWKHLQYLWURDQGLQYLYRVWXGLHVDQGWKHUHVXOWVRIZKLFKDUHGHVFULEHG KHUHLQ 7KHWDUJHWVSHFLILFLW\RI+X57I13ZDVFRQILQHGWR7IH[SUHVVLQJFDQFHUFHOOV $IWHUDFKLHYLQJVSHFLILFWDUJHWLQJDQGHIILFLHQF\LQ+X5JHQHNQRFNGRZQZHFRPSDUHG WKH HIILFLHQF\ RI +X57I13 LQ $DQG+&&OXQJFDQFHUFHOOVZLWKYDULHGOHYHOVRI 7I5 H[SUHVVLRQDQG05&OXQJILEUREODVWVZLWKQHJOLJLEOH7I5H[SUHVVLRQ7KHGRZQUHJXODWLRQRI

Ϯϯ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

+X5DQGFRUUHVSRQGLQJFHOOJURZWKSDWWHUQVZHUHDQDO\]HGIRUKDQGKDIWHU+X57I13RU &7I13FRQWUROVL51$>7I13@ WUHDWPHQWVDQGFRPSDUHGZLWKXQWUHDWHGFRQWUROV>)LJ$%@ $W ERWK  K DQG  K +X57I13 VLJQLILFDQWO\ VXSSUHVVHG WKH H[SUHVVLRQ RI +X5 P51$ >6XSSOHPHQWDU\)LJ6@DQGSURWHLQ>)LJ$6XSSOHPHQWDU\)LJV6DQG6@DFFRPSDQLHG E\FHOOJURZWKLQKLELWLRQ>)LJ%p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“ WKDQLQ Ϯϰ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

&7I13WUHDWHGFHOOV“p $WKWKLVGLIIHUHQFHZDVJUHDWHU+X57I13 “&7I13“p LQGLFDWLQJLQFUHDVHGFHOOJURZWKLQKLELWLRQRYHUWLPH $ VLPLODU WUHQG ZDV REVHUYHG LQ +&& FHOOV LQ ZKLFK WKH FHOO JURZWK LQ WKH +X57I13 WUHDWHGJURXS“ ZDVVLJQLILFDQWO\LQKLELWHGLQFRPSDULVRQZLWKWKH&7I13WUHDWHG JURXS“p DWK$WKWKLVGLIIHUHQFHZDVJUHDWHULQ+&&FHOOV+X5 7I13“&7I13“ &RQYHUVHO\QRVLJQLILFDQWGLIIHUHQFHZDVREVHUYHG EHWZHHQ &7I13 DQG +X57I13WUHDWHG 05& FHOOV DW HLWKHU  K RU  K LQGLFDWLQJ OHVV HIILFLHQW WDUJHWHG +X57I13EDVHG FHOO JURZWK LQKLELWLRQ +HQFH 7IPRGLILHG 13V FDUU\LQJ VL+X5 VKRZHG VHOHFWLYLW\ WRZDUGV 7I5RYHUH[SUHVVLQJ OXQJ FDQFHU FHOOV OHDGLQJ WR HIIHFWLYH JHQHNQRFNGRZQDQGFHOOJURZWKLQKLELWLRQ 

+X57I13LQGXFHV*FHOOF\FOHDUUHVWDQGDSRSWRVLVLQOXQJFDQFHUFHOOV 7KH SURJUHVVLRQ RI WKH FHOO F\FOH LQ $ +&& DQG 05& FHOOV DIWHU WUHDWPHQW ZLWK HLWKHU +X57I13 RU &7I13 IRU  K DQG  K ZDV HYDOXDWHG &RPSDUHG ZLWK FHOOV WUDQVIHFWHG ZLWK &7I13 D ODUJH LQFUHDVH LQ WKH * SRSXODWLRQ ZDV REVHUYHG LQ +X57I13 WUHDWHG$DQG+&&FHOOVDWKDQGKLQGLFDWLQJDSURQRXQFHGFHOOF\FOHDUUHVWDWWKH *SKDVH>)LJ&p@,QWHUHVWLQJO\QRVLJQLILFDQWFHOOJURZWKDUUHVWDWWKH*SKDVHZDV REVHUYHGLQ05&FHOOVXSRQ+X57I13WUHDWPHQWFRPSDUHGWR&7I13WUHDWPHQW>)LJ&@ +HQFHWKHSRVVLEOHXQGHUO\LQJPHFKDQLVPRI+X57I13LQGXFHGFHOOJURZWKLQKLELWLRQLQOXQJ FDQFHUFHOOVRFFXUVvia*FHOOF\FOHDUUHVW 2QH TXHVWLRQ WKDW DULVHV LV ZKHWKHU VL51$WDUJHWHG +X5 NQRFNGRZQ LQGXFHV RQO\ JURZWK DUUHVW RU VXEVHTXHQWO\ SURFHHGV WR DSRSWRWLF FHOO GHDWK 7R DQVZHU WKLV TXHVWLRQ ZH H[DPLQHGIRUFDVSDVHDQG3$53E\ZHVWHUQEORWWLQJDQGIRU$QQH[LQ9SRVLWLYHFHOOVE\IORZ Ϯϱ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

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p @ +X57I13 WUHDWPHQW DOVR UHVXOWHG LQ SRRU LQYDVLRQ RI $ FHOOV DV HYLGHQW IURPWKHPLFURVFRS\LPDJHVFRPSDUHGZLWK&7I13DQGXQWUHDWHGFRQWUROV>)LJ%p@ 6LJQLILFDQWO\IHZHUFHOOVLQYDGHGLQWKH+X57I13JURXSDVVKRZQLQWKHEDUGLDJUDPDWERWK KDQGK>p @

In vivoELRGLVWULEXWLRQDQGTXDQWLILFDWLRQRIVL51$FDUU\LQJ7I13 +DYLQJGHPRQVWUDWHGWKHin vitro HIILFDF\RI+X57I13RXUQH[WVWHSZDVWRVWXG\WKH GLVWULEXWLRQ EHKDYLRU RI 7I13 in vivo 7KH ELRGLVWULEXWLRQ RI 7I13 ZDV PRQLWRUHG LQ VXEFXWDQHRXV $ DQG +&& WXPRU PRGHOV ZLWK ,&*7I13 )LJ $ VKRZV WKH ,9,6Š VSHFWUXPOLYHLPDJHVIURPWXPRUEHDULQJPLFHWKDWZHUHLQWUDYHQRXVO\LY LQMHFWHGZLWK,&* Ϯϲ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

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in vivo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Ϯϳ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

UHVSHFWLYHO\ LQ $ DQG  DQG  UHVSHFWLYHO\ LQ +&& >)LJ % p @ $OWKRXJKVL51$OHYHOVGHWHFWHGDPRQJWKH+X57I13WUHDWHGVDPSOHVYDULHGQHYHUWKHOHVVWKH OHYHOVZHUHVLJQLILFDQWO\KLJKHUFRPSDUHGWR&7I13+HQFHLWLVHYLGHQWWKDW+X57I13ZDV FDSDEOHRIVXFFHVVIXOO\GHOLYHULQJLWVVL51$SD\ORDGWRWKHWDUJHWWLVVXHin vivo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p)LJ $@ /LNHZLVHWKHUHGXFWLRQVLQ+&&WXPRUYROXPHVZHUHDOVRKLJKO\SURQRXQFHGXSRQ +X57I13 WUHDWPHQW 7KH DYHUDJH WXPRU YROXPHV DW WKH WHUPLQDWLRQ RI WKH VWXG\ ZHUH  PP IRU &7I13WUHDWHG WXPRUV YHUVXV  PP  LQKLELWLRQ p   IRU +X57I13 WUHDWHG WXPRUV >)LJ %@ 7XPRU JURZWK ZDV FOHDUO\ LQKLELWHG XSRQ LQWUDWXPRUDO WUHDWPHQW LQGLFDWLQJ WKH HIILFDF\ RI +X57I13 0RUHRYHU WKH +X5 SURWHLQ H[SUHVVLRQ SDWWHUQ LQ WKH LVRODWHG$>)LJ$@DQG+&&>)LJ%@WXPRUWLVVXHVIURPPLFHWUHDWHGZLWK+X57I13 VKRZHGDPDUNHGUHGXFWLRQDIWHUWZRWUHDWPHQWVFRPSDUHGZLWKPLFHWUHDWHGZLWK&7I137KLV ILQGLQJVXJJHVWVWKDWWKHDQWLWXPRUDFWLYLW\ZDVFDXVHGE\+X5NQRFNGRZQin vivo 6LQFH LW FDQ EH DUJXHG WKDW LQWUDWXPRUDO WUHDWPHQWV DUH QRW FOLQLFDOO\ UHOHYDQW IRU OXQJ FDQFHUWUHDWPHQWZHQH[WDVVHVVHGWKHGHOLYHU\HIILFLHQF\DQGWKHWKHUDSHXWLFHIIHFWRIWDUJHWHG +X57I13XSRQLYDGPLQLVWUDWLRQLQPLFHEHDULQJ$OXF64WXPRUV$VVKRZQLQ)LJ& WXPRUJURZWKZDVVLJQLILFDQWO\LQKLELWHGXSRQ+X57I13WUHDWPHQWp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Ϯϵ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

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p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p @ )XUWKHUPRUH+X57I13WUHDWHGWXPRUVVKRZHGLQFUHDVHG DFWLYDWLRQ RI FDVSDVH DQG 3$53 FRPSDUHG WR &7I13WUHDWHG WXPRUV >6XSSOHPHQWDU\ )LJ 6@ $ VLPLODU VWXG\ FRQGXFWHG ZLWK PLFH EHDULQJ VXEFXWDQHRXV +&& WXPRUV GHPRQVWUDWHG WKH HIILFLHQF\ RI LY DGPLQLVWHUHG +X57I13 LQ WDUJHWHG DQWLWXPRU DFWLYLW\ ϯϬ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

PHGLDWHGE\VL+X5JHQHNQRFNGRZQ7KHLYDGPLQLVWUDWLRQRI+X57I13UHVXOWHGLQVOXJJLVK WXPRU JURZWK FRPSDUHG ZLWK WKH DFWLYH JURZWK REVHUYHG LQ &7I13WUHDWHG WXPRUV >)LJ (@ 7KHWXPRUYROXPHVZHUHPPIRUWKH+X57I13WUHDWHGJURXSDQGPPIRUWKH& 7I13WUHDWHGJURXSLQKLELWLRQp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± WUHDWHG WXPRUV $ DQG +&&  WKDQ LQ WKH +X57I13WUHDWHG WXPRUV >6XSSOHPHQWDU\ )LJV 6  6@ 7KHVH REVHUYDWLRQV VXJJHVW WKDW +X5 H[SUHVVLRQ LV FORVHO\ DVVRFLDWHG ZLWK FHOOXODUSUROLIHUDWLRQ,WFRQILUPVRXUHDUOLHUGDWDWKDWLQKLELWLRQRI+X5H[SUHVVLRQDEURJDWHVWKH WXPRUSUROLIHUDWLRQUDWH$UDSLGO\JURZLQJWXPRURIWHQH[SUHVVHV&'DPLFURYHVVHOGHQVLW\ 09' PDUNHUZKLFKLVGHWHFWDEOHE\LPPXQRKLVWRFKHPLVWU\>@:HGHWHFWHGVLJQLILFDQW ϯϭ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

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in vivo ,Q RUGHU WR LQYHVWLJDWH WKH DQWLWXPRU DFWLYLW\ RI +X57I13 RQ OXQJ PHWDVWDVLV ZH PRQLWRUHG WKH WXPRU JURZWK LQ PLFH EHDULQJ $OXF OXQJ PHWDVWDVLV IROORZLQJ +X57I13 WUHDWPHQW$VVKRZQLQ)LJ$WKHGHFD\RIELROXPLQHVFHQFHVLJQDOVIURP+X57I13WUHDWHG $OXFOXQJPHWDVWDVHVZHUHGLVWLQFWO\YLVLEOHHVSHFLDOO\DWWKHWKDQGWKZHHNVLQGLFDWLQJD VLJQLILFDQWO\ UHGXFHG WXPRU EXUGHQ FRPSDUHG ZLWK &7I13WUHDWHG JURXSV  LQKLELWLRQ DW ZHHN  p    7KH DYHUDJH UDGLDQFH RI WKH +X57I13WUHDWHG JURXSV ZDV UHGXFHG RU XQFKDQJHG FRPSDUHG ZLWK WKH HQKDQFHG UDGLDQFH REVHUYHG LQ WKH &7I13WUHDWHG JURXSV $QDO\VLVRIWKZHHNLPDJHVVKRZHGDQDYHUDJHUDGLDQFHRISVHFFPVU IRU&7I13 ZKLFK LV D IROG LQFUHDVH RYHU +X57I13WUHDWHG WXPRU VLJQDOV RI  SVHFFPVU p  $VH[SHFWHGWKHUHZDVOHVV+X5SURWHLQH[SUHVVLRQLQWKHH[FLVHGOXQJWLVVXHIURPWKH +X57I13WUHDWHGJURXSWKDQLQOXQJWLVVXHIURPWKH&7I13WUHDWHGJURXS>)LJ%@$VVKRZQ ϯϮ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

LQ )LJ % WKH PHWDVWDWLF QRGXOHV LQ +X57I13WUHDWHG OXQJV   LQKLELWLRQ  ZHUH VLJQLILFDQWO\ UHGXFHG ZKHUHDV WXPRU QRGXOHV RFFXSLHG D PDMRU SRUWLRQ RI WKH &7I13 WUHDWHG OXQJV  p   ,+& DQDO\VLV RI WKH OXQJ OREHV UHYHDOHG D VLJQLILFDQW XS WR WKUHHIROG UHGXFWLRQLQWKH+X5SURWHLQOHYHOVLQ+X57I13WUHDWHGJURXSVFRPSDUHGZLWK&7I13WUHDWHG JURXSV>)LJ& p@ &RUUHVSRQGLQJO\ WKHUH ZDV D WZRIROG UHGXFWLRQ LQ 09' LQGLFDWHG E\ &' H[SUHVVLRQ LQVHFWLRQVRIOXQJOREHVIURP+X57I13WUHDWHGDQLPDOVp 6LPLODUO\.L SRVLWLYH FHOOV ZHUH UHGXFHG WKUHHIROG LQ +X57I13WUHDWHG JURXSV LQGLFDWLQJ VLJQLILFDQWO\ OHVVSUROLIHUDWLRQFRPSDUHGZLWKWKH&7I13WUHDWHGJURXSp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

ϯϯ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

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in vitro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n vivoELRGLVWULEXWLRQVWXG\GHPRQVWUDWHGSUHIHUHQWLDODFFXPXODWLRQRI'\+X57I13 LQWXPRUPLOLHXDQGWKLVUHVXOWFRUUHODWHGZLWK,&*7I13GLVWULEXWLRQLQWXPRUV6LQFHQHXURQV DQGEUDLQFDSLOODU\HQGRWKHOLDOFHOOVLQKXPDQDQGPLFHDUHNQRZQWRH[SUHVV7I5LQVLJQLILFDQW ϯϰ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

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via V\VWHPLF URXWH DGPLQLVWUDWLRQ ZDV VLJQLILFDQW VLQFH WXPRU YROXPH LQKLELWLRQ ZDV FRUUHODWHG ZLWK +X5 NQRFNGRZQ 7KH SUROLIHUDWLYHQDWXUHDQGDFWLYHYDVFXODWXUHIRUPDWLRQZHUHVLJQLILFDQWO\OHVVLQ+X57I13WUHDWHG WXPRUV DV DQDO\]HG E\ ,+& VWDLQLQJ 7KH PHFKDQLVP E\ ZKLFK +X57I13 UHGXFHV WXPRU ϯϱ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

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in vivo DQGFRQFXUZLWKRXUSUHYLRXV in vitro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ϯϲ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

LPPXQHV\VWHPVLQFH+X5KDVSUHYLRXVO\EHHQUHSRUWHGWRSOD\DUROHLQLQIODPPDWLRQ>@ )XUWKHU UHFHQW UHSRUWV RQ WKH UROH RI 3'/3' LQ VXSSUHVVLQJ KRVW LPPXQH 7FHOOV EHJV WKH TXHVWLRQLI+X57I13WUHDWPHQWZLOOLPSDFWWKH3'/3'VLJQDOLQJDQGDOWHUWKHKRVWLPPXQH V\VWHPWRHQKDQFHWKHDQWLWXPRULPPXQHDFWLYLW\7KHVHTXHVWLRQVZKLOHRILQWHUHVWDUHEH\RQG RIWKHVFRSHRIWKHSUHVHQWVWXG\EXWZDUUDQWLQYHVWLJDWLRQLQWKHIXWXUH>@ ,QFRQFOXVLRQ ZHKDYHGHPRQVWUDWHG+X57I13WUHDWPHQWHIIHFWLYHO\LQKLELWVOXQJFDQFHUJURZWKERWKin vitro DQGin vivo                 ϯϳ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

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

ϯϴ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted HuR RNAi nanoparticle therapy for lung cancer 

  5HIHUHQFHV 

&KHYDOLHU7/1RQVPDOOFHOOOXQJFDQFHUWKHFKDOOHQJHVRIWKHQH[WGHFDGHFront Oncol 



$OOHQ 70 /LJDQGWDUJHWHG WKHUDSHXWLFV LQ DQWLFDQFHU WKHUDS\. Nat Rev Cancer : 



%DH ,QWHUQHW@ -DQGLDO5HG/DQGHV%LRVFLHQFH$XVWLQ



0L\DWDSVFPð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p

p   

ϱϬ  Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on June 1, 2017; DOI: 10.1158/1535-7163.MCT-17-0134 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-targeted nanoparticle delivery of HuR siRNA inhibits lung tumor growth in vitro and in vivo by disrupting the oncogenic activity of the RNA-binding protein HuR Ranganayaki Muralidharan, Anish Babu, Narsireddy Amreddy, et al. Mol Cancer Ther Published OnlineFirst June 1, 2017.

Updated version Supplementary Material Author Manuscript

E-mail alerts Reprints and Subscriptions Permissions

Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-17-0134 Access the most recent supplemental material at: http://mct.aacrjournals.org/content/suppl/2017/06/01/1535-7163.MCT-17-0134.DC1 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected].

Downloaded from mct.aacrjournals.org on August 17, 2017. © 2017 American Association for Cancer Research.