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Title In vitro optimization of 2 -OMe-4 -thioribonucleoside-modified anti-microRNA oligonucleotides and its targetingdelivery to mouse liver using a liposomal nanoparticle

Author(s) Takahashi Mayumi Yamada Naoki Hatakeyama Hiroto Murata Manami Sato Yusuke Minakawa NoriakiHarashima Hideyoshi Matsuda Akira

Citation Nucleic acids research 41(22) 10659-10667httpsdoiorg101093nargkt823

Issue Date 2013-12

Doc URL httphdlhandlenet211554780

Type article

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Hokkaido University Collection of Scholarly and Academic Papers HUSCAP

In vitro optimization of 20-OMe-40-thioribonucleosidendashmodified anti-microRNAoligonucleotides and its targeting deliveryto mouse liver using a liposomal nanoparticleMayumi Takahashi1 Naoki Yamada1 Hiroto Hatakeyama1 Manami Murata1

Yusuke Sato1 Noriaki Minakawa2 Hideyoshi Harashima1 and Akira Matsuda1

1Faculty of Pharmaceutical Sciences Hokkaido University Kita-12 Nishi-6 Kita-ku Sapporo 060-0812Japan and 2Graduate School of Pharmaceutical Sciences The University of Tokushima Shomachi 1-78-1Tokushima 770-8505 Japan

Received July 14 2013 Revised August 20 2013 Accepted August 21 2013

ABSTRACT

MicroRNAs (miRNAs) are small noncoding RNAsthat regulate gene expression post-transcriptionallyPrevious studies which characterized miRNAfunction revealed their involvement in fundamentalbiological processes Importantly miRNA expressionis deregulated in many human diseases Specificinhibition of miRNAs using chemically modified anti-miRNA oligonucleotides (AMOs) can be a potentialtherapeutic strategy for diseases in which a specificmiRNA is overexpressed 20-O-Methyl (20-OMe)-40-thioRNA is a hybrid type of chemically modifiedoligonucleotide exhibiting high binding affinityto complementary RNAs and high resistanceto nuclease degradation Here we evaluate 20-OMe-40-thioribonucleosides for chemical modification onAMOs Optimization of the modification pattern usinga variety of chemically modified AMOs that areperfectly complementary to mature miR-21 revealedthat the uniformly 20-OMe-40-thioribonucleosidendashmodified AMO was most potent Further investiga-tion showed that phosphorothioate modificationcontributed to long-term miR-122 inhibition bythe 20-OMe-40-thioribonucleosidendashmodified AMOMoreover systemically administrated AMOs tomouse using a liposomal delivery system YSK05-MEND showed delivery to the liver and efficientinhibition of miR-122 activity at a low dose in vivo

INTRODUCTION

MicroRNAs (miRNAs) are a class of endogenouslyexpressed small noncoding RNAs (18 25 nt) which

regulate gene expression post-transcriptionally Inanimals single-stranded mature miRNAs hybridize tothe 30 untranslated region (30UTR) of the target mRNAthrough complete base paring with positions 2ndash8 of themiRNA known as the seed region Binding on the seedregion nucleates miRNAndashmRNA association and causestranslational inhibition or mRNA degradation (12)Because complementarity of the seed region consists ofonly 7 nt a single miRNA may regulate multiple genesand a single mRNA can be modulated by several differentmiRNAs (3ndash5) To date gt1000 miRNAs have beenidentified in humans and regulate up to 60 of proteincoding genes (67) MiRNAs are implicated in importantfunctions in the biological process including cell differen-tiation proliferation development metabolism and apop-tosis Furthermore up- or downregulation of miRNAexpression is correlated with a variety of human diseasessuch as cancer viral infection and cardiovascular dis-orders (8) Thus regulation of specific miRNA functionis a promising therapeutic strategy for treatment of suchdiseasesAmong the approaches to modulate the function of

miRNAs anti-miRNA oligonucleotide (AMO)-based in-hibition has been most widely used not only to exploit thebiological function of miRNAs but also as candidates fortherapeutic agents (9) To develop oligonucleotide (ON)-based therapeutic strategies there are several issues toovercome namely poor stability of ONs in biologicalfluids weak binding affinity to target RNA poorcellular uptake and unfavorable immunostimulatoryactivity Thus far a wide variety of chemically modifiedONs have been developed to date including 20-O-methyl(20-OMe) (10ndash13) 20-O-methoxyethyl (14) and lockednucleic acid (LNA) to overcome these disadvantages(15ndash17) These chemical modifications have successfullybeen applied to an antisense technology as well as

To whom correspondence should be addressed Tel +81 11 706 3228 Fax +81 11 706 4980 Email matudapharmhokudaiacjp

Published online 11 September 2013 Nucleic Acids Research 2013 Vol 41 No 22 10659ndash10667doi101093nargkt823

The Author(s) 2013 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (httpcreativecommonsorglicensesby-nc30) which permits non-commercial re-use distribution and reproduction in any medium provided the original work is properly cited For commercialre-use please contact journalspermissionsoupcom

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ON-based therapeutic technologies (eg siRNAsaptamers ribozymes) Two successful reports of 20-OMendashmodified AMOs were reported in 2004 (1819)lending credence to the idea of using chemicallymodified AMOs Furthermore considerable efforts inoptimization have been dedicated to develop AMOs as anew therapeutic agent and several chemically modifiedAMOs are currently undergoing clinical trials (20)Therefore further optimization of chemical modificationsfor AMO-based miRNA suppression will continue toimprove this therapeutic approachWe developed a novel chemically modified ON 20-OMe-

40-thioRNA (21) (Figure 1) which can be considered ahybrid chemical modification based on 20-OMe RNA (13)and 40-thioRNA (22ndash28) In our previous study wereported the development of 20-OMe-40-thioribonucleosidendashmodified siRNA (29) Optimizationof both the number and position of modification by 20-OMe-40-thioribonucleoside afforded modified siRNAswith more potent and persistent RNAi activity Inaddition investigation of the duration of RNAi activityresulted in long-lasting gene silencing in vitro owing tothe improved intracellular stability of 20-OMe-40-thioribonucleosidendashmodified siRNA Like many otherchemically modified ONs that can successfully be appliedto AMO as well as siRNA we expected that 20-OMe-40-thioribonucleoside modification acts as a promising AMOTherefore we set out to evaluate the utility of 20-OMe-40-thioribonucleoside for chemical modification on AMOs Inthis study we investigated the modification pattern ofAMOs by 20-OMe-4-thioribonucleoside in terms ofpotency and duration of activity in two kinds of targetmiRNA (miR-21 and miR-122) in vitro Moreover system-ically administrated AMOs to mouse liver using aliposomal delivery system a multifunctional envelope-type nano device (MEND) with a pH-sensitive cationiclipid YSK05 (YSK05-MEND) (30) showed efficient in-hibition of miR-122 activity at a low dose in vivoimplicating potential use of 20-OMe-40-thioribonucleosidendashmodified AMO in nucleic acid therapy

MATERIALS AND METHODS

Oligonucleotides

The chemically modified AMOs used in this study weresynthesized on an Applied Biosystem 3400 DNA synthe-sizer according to our previous report (21) Thus supportbound chemically modified AMOs were synthesized usingthe corresponding phosphoramidite units at a 10mmolscale following the standard procedure described for

oligoribonucleotides Each of the phosphoramidite unitswas used at a concentration of 01M in dry acetonitrileand the coupling time was extended to 10min for eachstep AMOs with phosphorothioate (PS) backbone wereachieved by oxidation with 3H-12-benzodithiol-3-one-11-dioxide (Beaucage reagent) during ON synthesisAfter completion of the synthesis the CPG support wastreated with concentrated NH4OH or NH4OHEtOH (31)at 55C for 16 h In the case of CPG supports containingeither 20-F or 20-F-40-thioribonucleoside modificationthese were treated with methanolic ammonia (saturatedat 0C) at room temperature for 24 h Then the supportwas filtered off The filtrate was concentrated and the ONprotected by a DMTr group at the 50-end was chromato-graphed on a C-18 silica gel column with a linear gradientof acetonitrile (from 5 to 40) in 01 N TEAA buffer (pH70) The fractions containing the full-length ON werecombined and concentrated The residue was treatedwith aqueous acetic acid (70) for 20min at room tem-perature The solution was concentrated and the residuewas purified on reversed-phase high performance liquidchromatography using a Jrsquosphere ODS-M80 column(46 150mm YMC) with a linear gradient of acetonitrile(from 10 to 40) in 01 N TEAA buffer (pH 70) Thestructures of each RNA were confirmed by measure-ment of MALDI-TOFMASS spectrometry on UltraflexTOFTOF (Buruker Daltonics) The analytical dataof synthetic AMOs are summarized in SupplementaryTable S1

Tm measurement

Thermally induced transitions were monitored at 260 nmon a Beckman DU 650 spectrophotometer Samples wereprepared as follows AMO and target miRNA (3 mM each)were mixed in a phosphate buffer (10mM pH 70) con-taining 01mM EDTA and 1mM NaCl heated at 90Cfor 5min cooled gradually to room temperature and usedin thermal denaturation studies The sample temperaturewas increased 05Cmin

Cell culture

HeLa cells and Huh-7 cells were cultured at 37C under5 CO2 in Dulbeccorsquos modified Eaglersquos medium(DMEM Gibco) supplemented with 10 fetal bovineserum (FBS Thermo Fisher Scientific) 100 unitsml1

penicillin 100 mgml1 streptomycin respectively Cellswere regularly passaged to maintain exponential growth

Construction of miRNA reporter plasmids and luciferasereporter assay

The miRNA reporter plasmids were generated by cloningthe synthetic 50-phosphorylated ONs corresponding totandem perfect-match target sited for human miR-21 ormiR-122 into the 30UTR of firefly luciferase gene (luc2)in the pmirGLO Vector (Promega) HeLa cells or Huh-7cells were plated into 96-well plates at 10 000 cellswell inDMEM supplemented with 10 FBS 100 unitsml1

penicillin 100 mgml1 streptmycin After 24 h ofplating reporter plasmid and AMOs were co-transfectedinto the cells in triplicate using LipofectamineTM 2000

Figure 1 Structure of 20-modified-40-thioribonucleoside

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(Invitrogen) according to the manufacturerrsquos instructionsin 100 mlwell Opti-MEM (Invitrogen) AMO concentra-tions varied as indicated whereas plasmid concentrationsremained constant at 01 mgwell After 6 h of transfec-tion cells were re-fed with complete media Cells werelysed at indicated hours after transfection and luciferaseactivities were measured using the Dual-LuciferaseReporter Assay System (Promega) Relative fireflyluciferase signals were normalized against Renillaluciferase signals Results are expressed as relative FlucRluc ratios to that of mirGLO-treated cells Each experi-ment was performed at least three times

Preparation of AMO encapsulated liposome

As a liposomal delivery system YSK05-MENDs encap-sulating AMOs were prepared by a t-BuOH dilution pro-cedure (3031) Thus each AMO was mixed with 90(vv) t-BuOH containing YSK05 (synthesized as previ-ously described) (30) cholesterol (Avanti Polar Lipid)12-dimyristoyl-sn-glycerol and methoxyethyleneglycol2000 ether (Avanti Polar Lipid) at a molar ratio of70303 in 20mM citrate buffer (pH 40) at AMOlipidsof 01 (wtwt) under strong agitation to a t-BuOH concen-tration of 60 (vv) Then lipidsAMO mixture wasadded into 20mM citrate buffer (pH 40) under strongagitation to a t-BuOH concentration of lt12 (vv)Ultrafiltration was performed to remove t-BuOHreplace external buffer with phosphate buffered saline(PBS pH 70) and concentrate a resulting YSK05-MEND encapsulating AMO (AMO-YSK05-MEND)The average diameter and zeta-potential of the AMO-YSK05-MENDs were determined using a ZetasizerNano ZS ZEN3600 (MALVERN InstrumentWorchestershire)

In vivo experiments

Female BALBc mice (8 weeks old) were purchased fromJapan SLC All in vivo experiments were approved by theInstitutional Animal Care and Use Committee One daybefore the administration serum was collected formeasuring cholesterol concentration Each AMO-YSK05-MEND was diluted to the appropriate concentra-tions in PBS (pH 74) followed by intravenous adminis-tration via the tail vein at a dose of 1mg AMOkg in10 15mlkg given once a day every other day forthree times At 48 h after the last injection liver andblood were collected Blood sample was centrifuged at8g at 4C for 5min to obtain plasma To obtain serumblood sample was stored overnight at 4C followed bycentrifugation (10 000 rpm 4C 10min) Cholesterol con-centration in plasma and alanine aminotransferase level inserum were determined using a cholesterol E-test WAKOand Transaminase CII-test WAKO (Wako) according tothe manufacturerrsquos protocols Total RNAs in liver wereisolated using TRIzol (Invitrogen) according to the manu-facturerrsquos protocols Then isolated RNAs were reversetranscribed using a High Capacity RNA-to-cDNA kit(Applied Biosystems) according to manufacturerlsquosprotocol A quantitative polymerase chain reaction(PCR) analysis was performed with 20 ng of cDNA

using Fast SYBR Green Master Mix (AppliedBiosystems) on the Lightcycler480 System II (RocheApplied Science) All reactions were performed at avolume of 15 ml The primers for qRT-PCR are asfollows mouse AldoA (forward) 50-GATGGGTCCAGCTTCAAC-30 and (reverse) 50-GTGCTTTCCTTTCCTAACTCTG-30 mouse Bckdk (forward) 50-AGGACCTATGCATGGCTTTG-30 and (reverse) 50-CCGTAGGTAGACATCCGTG-30 mouse Ndrg3 (forward) 50-ATGGGCTACATACCATCTGC-30 and (reverse) 50-TCTGACTGATTGCTGGTCAC-30 mouse Hprt1 (forward) 50-CGTGATTAGCGATGATGAAC-30 and (reverse) 50-GCAAGTCTTTCAGTCCTGTC-30 Each data was normalized by Hprt1expression All experiments were performed in triplicateand data show mean values from at least three assays

Statistical analysis

Comparisons between multiple treatments were madeusing one-way analysis of variance (ANOVA) followedby the SNK test Pair-wise comparisons between treat-ments were made using a studentrsquos t-test Plt 005 wasconsidered significantly different

RESULTS AND DISCUSSION

Evaluation of inhibitory activity of AMOs against miR-21

We first synthesized modified AMOs against miR-21 amiRNA that is overexpressed in many tumors and thus isconsidered to be a potential therapeutic target in oncology(32) The sequences and modification patterns of AMOswere shown in Table 1 Because 20-F-40-thioRNA showedhighest hybridization ability with its complementary RNAamong the chemically modified ONs tested (2133) wesynthesized chimeric 20-OMe-40-thio20-F-40-thiondashmodifiedAMOs (AM21SMF1 and AM21SMF2) along with20-OMe-40-thiondashmodified AMO (AM21SM) which arecomplementary and the same length (22-mer) as maturemiR-21 For comparison 40-oxo congeners (AM21MAM21MF1 and AM21MF2) were also prepared BecauseHutvagner et al reported that AMOs possessing a comple-mentary sequence core with 5 nt flanking sequences onboth 50- and 30-ends showed more potent anti-miRNAactivity (19) we also designed a 32-mer uniformly 20-OMe-40-thioribonucleosidendashmodified AMO (AM21SM-L) which exhibits perfect complimentarity to miR-21 and5 nt flanking regions on both ends as well as that of20-OMe (AM21M-L)To evaluate anti-miRNA activity of the synthetic

AMOs we constructed a miR-21 luciferase reporterplasmid (34) which has two perfectly matched miR-21binding sites arranged in tandem on 30UTR of fireflyluciferase gene (pmirGLO21) The firefly luciferaseactivity was completely suppressed (37) when thepmirGLO21 was transfected into HeLa cells a cell lineknown to have high endogenous levels of miR-21(Figure 2) The activity of pmirGLO which has nomiR-21 binding sites was high (normalized as 100) ontransfection into HeLa cells Our hypothesis is that intro-duction of miR-21 AMO can prevent miR-21 binding tothe pmirGLO21 target sites resulting in increased

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luciferase expression by either causing translational inhib-ition or cleavage of the mRNA We then tested anti-miRNA activity of AMOs As shown in Figure 2 allour AMOs showed miR-21 inhibitory activity in a dose-dependent manner In general 40-thiondashmodified AMOs(AM21SM AM21SMF1 and AM21SMF2) showedmuch higher activity than 40-oxo congeners (AM21MAM21MF1 and AM21MF2) The activities of chimeraAMOs (AM21SMF1 AM21SMF2 AM21MF1 andAM21MF2) were less effective compared with uniformlymodified AMOs (AM21SM and AM21M) Contrary tothe results reported by Hutvanger et al (19) no significantimprovement was observed in 32-mer AM21M-L (300at 50 nM) compared with 22-mer AM21M (279 at50 nM) Furthermore among the AMOs tested theshorter AMO namely AM21SM was more potent thanAM21SM-L (743 and 459 respectively) in the case of40-thiondashmodified AMOs The highest activity was

observed with AM21SM with a luciferase level of743 at 50 nM which was gt25 times as much as thatof AM21M (279 at 50 nM)

It is known that there is a good correlation between Tm

value of AMOs and in vitro potency (35) Therefore wecompared the thermal stability of the AMOs with targetmiR-21 based on their Tm values As can be seen inTable 1 the Tm values of chimera AMOs which containeither 20-F or 20-F-40-thioribonucleoside modificationwere higher than those of the uniformly modifiedAMOs Although AM21SMF2 showed the highest Tm

value its anti-miR-21 activity was lowest among 40-thiondashmodified AMOs A similar trend was observed in a seriesof AMOs consisting of 40-oxo congeners Thus no correl-ation was observed between activity and binding affinityin our case Meanwhile as we reported previously (21) 20-OMe-40-thioRNA showed higher nuclease stabilitycompared with 20-OMe RNA 20-F-40-thioRNA and 20-FRNA Therefore chimera AMOs might be more suscep-tible to degradation than the uniformly modified AMOsserving as a potential explanation why AM21SM showedthe highest activity amongst all AMOs tested From theseresults it can be concluded that the 20-OMe-40-thioribonucleoside modification is useful for inhibitionof miRNA function Also uniform modification issimple and seems to be the most promising choice forsubsequent studies

Improvement in duration of anti-miRNA activity ofAMOs by backbone substitution

To examine if 20-OMe-40-thioribonucleosidendashmodifiedAMO is capable of inhibiting other miRNAs we chosemiR-122 as another miRNA target MiR-122 is a liver-specific miRNA known to be involved in cholesterolmetabolism fatty acid metabolism (36) and hepatitis Cvirus replication (37) Many successful studies of miR-122 inhibition by AMOs both in vitro and in vivo havebeen reported (2036ndash42) and some of them are cur-rently under investigation in clinical trials (20) Wehypothesized that 20-OMe-40-thioribonucleosidendashmodifiedAMO could have high potency to inhibit miR-122 as wellas miR-21

We synthesized a uniformly 20-OMe-40-thioribonucleosidendashmodified AMO which is complemen-tary to and the same length (23-mer) as mature miR-122

Table 1 Sequence and modification patterns of anti-miR-21 AMOs

AMO Sequencea Tm (C)b

AM21SM 50-UCAACAUCAGUCUGAUAAGCUA-30 641AM21SMF1 50-ucAAcAucAGucuGAuAAGcuA-30 692AM21SMF2 50-UCaacaucagucugauaagcUA-30 711AM21M 50-UCAACAUCAGUCUGAUAAGCUA-30 599AM21MF1 50-ucAAcAucAGucuGAuAAGcuA-30 642AM21MF2 50-UCaacaucagucugauaagcUA-30 679AM21SM-L 50-UCUUAUCAACAUCAGUCUGAUAAGCUAACCUU-30 621AM21M-L 50-UCUUAUCAACAUCAGUCUGAUAAGCUAACCUU-30 580

aUppercase letters represent 20-OMe lowercase letters represent 20-F bold uppercase letters are 20-OMe-40-thioribonu-cleotides bold lowercase letters are 20-F-40-thioribonucleotides bTms were measured versus with target miR-21 (22-mer)in a phosphate buffer (10mM pH 70) containing 01mM EDTA and 1mM NaCl 3 mM strand concentration Valueswere given as an average of three independent experiments

Figure 2 Anti-miR-21 activity of modified AMOs HeLa cells wereco-transfected with the miR-21 reporter plasmid (mirGLO21) and theindicated modified anti-miR-21 AMOs at the indicated concentrationsThe dual luciferase assay was performed at 24 h after transfection Dataare shown as mean with standard deviation (SD)

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with unmodified phosphodiester (PO) backbone(AM122SM Table 2) Because ONs can be degraded bynuclease by cleaving a phosphodiester linkage in biologicalfluid substitution of a PO linkage for a PS linkage is idealto prevent such nuclease cleavage reactions In fact PSmodification has successfully been applied to many previ-ously reported AMOs Because our end goal of this study isin vivo application we synthesized 20-OMe-thioribonucleosidendashmodified AMOs with PS backbones(AM122SM-PS) For comparison we also synthesized uni-formly 20-OMendashmodified AMOs with either PO or PSbackbones (AM122M and AM122M-PS respectively)Recently Obad et al developed seed targeting 8-mer tinyLNAs which can simultaneously inhibit miRNA familiesthat share the same seed region (42) According to thisreport we also prepared a seed targeting 8-mer(AM122SM-PS 8 nt) along with two shorter sequences(AM122SM-PS 15 nt and AM122SM-PS 20 nt) toexamine their potency as well as to find an optimal lengthof AMOs

Each AMO and a miR-122 reporter plasmid (pmirGLO122) constructed in a same manner to pmirGLO21 wereco-transfected into Huh-7 cells at various AMO concen-trations (05ndash5 nM) At 24 h after transfection we har-vested the cells and carried out dual luciferase reporterassay The resulting AMO activities were shown inFigure 3 As was the case of miR-21 inhibition allAMOs showed miR-122 inhibitory activity in a dose-dependent manner and AM122SM gave higher activitythan AM122M (639 versus 366 at 5 nM) No obviousdifference was observed in the activity between AMOswith PO and PS backbones Concerning of the length ofAMO anti-miRNA activity decreased as AMO lengthbecame shorter and tiny AMO namely AM122SM PS8nt did not show any activity Thus the 20-OMe-40-thioribonucleosidendashmodified AMO possessing comple-mentary matched sequence and length seems to besuitable for miRNA inhibition

In our previous study we showed prolonged activityof 20-OMe-40-thioribonucleosidendashmodified siRNA (29)Thus we expected that the 20-OMe-40-thioribonucleosidemodification can prolong the activity of AMOs We nextperformed a time-course experiment As described aboveno difference between PO and PS AMOs was observed inthe activity after 24 h However considerable differences

were observed after 48 h where the activities of PO AMOs(AM122SM and AM122M) declined (Figure 4) whileactivities of PS AMOs (AM122SM-PS and AM122M-PS) increased AM122SM-PS showed dramatic increaseof the activity throughout the assay as much as 90at 72 h From these results it can be concluded that 20-OMe-40-thioribonucleosidendashmodified AMO are superiorcompared with 20-OMendashmodified AMO and that PSmodification conferred long-term activity on AM122SMIn the case of the miR-21 study described above we

suggested that nuclease resistance is more closely relatedto AMO potency than the Tm values We wished toexplore further which of these two factors correlatedmore strongly with the potency To explore a correlationbetween the potency and physical properties of 20-OMe-40-thioribonucleotidendashmodified miR-122 AMOs in moredetail we first measured the Tm values of duplexes witha target miR-122 As can be seen in Table 2 AM122M

Table 2 Sequence and modification pattern of anti-miR-122 AMOs

AMO Sequencea Tm (C)b

AM122SM 50-ACAAACACCAUUGUCACACUCCA-30 658AM122SM-PS 50-ACAAACACCAUUGUCACACUCCA-30 648AM122SM-PS 20 nt 50-AAACACCAUUGUCACACUCC-30 607AM122SM-PS 15 nt 50-CCAUUGUCACACUCC-30 546AM122SM-PS 8 nt 50-CACACUCC-30 363AM122M 50-ACAAACACCAUUGUCACACUCCA-30 629AM122M-PS 50-ACAAACACCAUUGUCACACUCCA-30 580

aUpper case letters represent 20-OMe bold upper case letters are 20-OMe-40-thioribonucleotides underlined are PSbackbone modification bTms were measured versus miR-122 in a phosphate buffer (10mM pH 70) containing01mM EDTA and 1mM NaCl 3 mM strand concentration Values were given as an average of three independentexperiments

Figure 3 Anti-miR-122 activity of the modified AMOs Huh-7 cellswere co-transfected with the miR-122 reporter plasmid (mirGLO122)and the anti-miR-122 AMOs at the indicated concentrations The dualluciferase assay was performed at 24 h after transfection Data areshown as mean with SD

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had a Tm value of 629C and that of AM122M-PS waslower (Tm=580C and Tm=49C) On the otherhand AM122SM showed a higher Tm value (658C)compared with that of AM122M and AM122SM-PShad the Tm value of 648C with only a slight decreasefrom that of AM122SM (Tm=10C) It is wellknown that the PS modification lowers the Tm of ONs(05ndash07C per modification) (4344) This decrease ofthe binding affinity would cause loss of inhibitoryactivity of AMOs However replacement of the PObackbone of AM122SM with a PS had no effect on thebinding affinity of the AMO a possible explanation forthe retained AMO activityWe then investigated the nuclease stability of the

modified AMOs A series of AMOs were labeled at the50-end with 32P and incubated in 50 human plasmaThe reactions were analyzed by denaturing PAGE(Supplementary Figure S1) As expected PS AMOs ex-hibited higher stability than that of PO AMOs and aswe reported previously 20-OMe-40-thioribonucleosidendashmodified AMOs were slightly more stable than that ofthe corresponding 20-OMendashmodified AMOs It is worthnoting that AM122SM-PS was totally intact under theseconditions while AM122M-PS showed some mild degrad-ation The rank order of stability in plasma wasAM122SM-PSgtAM122M-PSgtAM122SMgtAM122Mindicating that AM122SM-PS is extremely stable againstnuclease degradation in human plasma These results sug-gested that the Tm value of the AMO had relatively littleimpact on AMO potency whereas nuclease resistanceseemed to have much greater effectFor therapeutic use nuclease resistance in biological

fluid tissues and cells is a prerequisite feature of chem-ically modified ONs Our previous comprehensive studyof nuclease stability revealed that 20-OMe-40-thioRNAwas significantly stable against both exo- and endonucle-ases in spite of consisting of PO backbones (21)Here we showed PS modification on the 20-OMe-40-thioribonucleosidendashmodified AMO can further improve

its nuclease resistance As a more specific scenario theendonuclease activity of Ago2 cleaves the unselectedstrand of RNA duplexes loading into the RISC It isthought that AMOs act primarily by hybridizing withmature miRNAs that have been loaded into the RISCWang et al explained the molecular mechanisms of targetRNA cleavage by a crystal structure of Ago protein (45)They found that both 20-OMe and PS substitution at thecleavage site (positions 100ndash110) disrupted Ago sliceractivity owing to configurations of the sulfur atoms inAgo protein Thus we suggest that the potency and long-term activity of the AM122SM-PS would be due in partto its resistance to cleavage by Ago Also PS modificationcould increase intracellular stability of AMOs conse-quently anti-miRNA activity AMOs increased over time(Figure 4) Taken together combinatorial use of 20-OMe-40-thioribonucleosides and a PS backbone is an attractivechoice for therapeutic AMOs

Targeted delivery and anti-miR-122 activity of 20-OMe-4-thioribonucleosidendashmodified AMO in mouse liver

In vitro studies showed that AM122SM-PS possessed themost favorable properties Hence we next carried outin vivo studies to assess whether AM122SM-PS couldalso inhibit miR-122 in mice

Two aspects of successful nucleic acid therapeutics aredelivery followed by ON stability We have developed anew liposomal nucleic acid delivery system YSK05-MEND which contains a pH-sensitive cationic lipid forefficient release of siRNAs from the endosome into thecytoplasm (29) Thus we prepared YSK05-MEND forin vivo delivery of AMOs to the liver YSK05-MENDsencapsulating AM122M-PS and AM122SM-PS repre-sented comparable size charge polydispersity and encap-sulation efficiency (Supplementary Table S2) We assessedin vivo efficacy of the modified miR-122 AMOs by treatingmice three times with intravenous injection of 1mgkgYSK05-MEND formulated AMO122SM-PS orAMO122M-PS every 2 days

As described above a single miRNA may control thelevels of multiple mRNAs (3ndash6) MiR-122 is no exceptionas many mRNAs whose expressions are directlycontrolled by miR-122 have been identified Elmen et aldemonstrated inhibition of miR-122 using LNA-modifiedAMOs LNA-antimiR (41) In their experiments dere-pressions of four mRNAs AldoA Bckdk Ndrg3 andCd320 all of which are direct targets of miR-122 in themouse liver were observed after treatment of mice withLNA-antimiR Therefore we conducted expressionanalysis of three miR-122 target mRNAs in the mouseliver AldoA Bckdk and Ndrg3 (Figure 5A) by real-time PCR 48 h after the last injection The levels of allthree mRNAs were higher in the AMO-treated micecompared with those treated with saline It is worthnoting that AM122SM-PS induced higher mRNA expres-sion levels than AM122M-PS in all three mRNAsexamined We also observed statistically significantdifferences between AM122SM-PS and AM122M-PS inthe expressions of both AldoA and Bckdk (Plt 005)

We next examined the change in plasma cholesterollevel associated with the increase in expression of these

Figure 4 Duration of inhibition by anti-miR-122 AMOs Huh-7 cellswere co-transfected with the miR-122 reporter plasmid (mirGLO122)and the anti-miR-122 AMOs at 5 nM The dual luciferase assay wasperformed at the indicated hours Data are shown as mean with SD

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mRNAs There was no obvious difference between theAMO- and saline-treated mice at 1 day after the lastdose while drastic changes were observed at day 6where the serum cholesterol levels were reduced by577 for AM122SM-PS and 525 for AM122M-PS(Figure 5B) without any hepatotoxicity (SupplementaryFigure S2) These results suggest that AM122SM-PS wasproperly delivered to the mouse liver by YSK05-MENDand elicited an anti-miR-122 effect

In the previous reports (2036ndash3840ndash42) AMOs havebeen administered either as nakedsaline formulated oras small molecule conjugates (eg cholesterol a cell-penetrating peptide) to assist in vivo delivery Theseapproaches often required relatively high doses (rangingfrom 10 to 80mgkg for mice) Instead our YSK05-MEND dosing strategy achieves efficient miR-122AMO delivery to the liver Although the mouse strains

used for our experiments versus other researchersrsquoprevious studies were different (eg we used inbredstrain mice BALBc which are considered to be genetic-ally identical whereas others used outbred strains suchas NMRI) we successfully increased miR-122 targetmRNA expression followed by a decrease in serum chol-esterol level with three intravenous doses of 1mgkgAM122SM-PS in mice

CONCLUSION

We demonstrated the inhibition of miRNAs with 20-OMe-40-thioribonucleosidendashmodified AMOs in vitro as wellas in vivo We first evaluated a variety of chemicallymodified AMOs that are complementary to maturemiR-21 and found the potency of the uniformly 20-OMe-40-thioribonucleosidendashmodified AMO to be the

Figure 5 Inhibition of miR-122 in mice (A) Levels of miR-122 target genes in liver RNA analyzed by qRT-PCR Each mRNA level was normalizedto the mean of saline control group Data are shown as mean with SD (n=4 48 h) (B) Plasma cholesterol levels in mice The data were normalizedto the mean of saline control group at each time point (Day 1 and Day 6) Data are shown as mean with SD (n=4) Plt 005 Plt 001 versussaline Plt 005 Plt 001 determined by one-way ANOVA followed by SNK test

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best in our series Further investigation revealed that PSmodification contributes to long-term miR-122 inhibitionby the 20-OMe-40-thioribonucleosidendashmodified AMOFor in vivo studies we took advantage of an efficient

delivery technology YSK05-MEND and successfullyincreased three miR-122 target mRNA levels in theAMO-treated mice liver followed by a decline of serumcholesterol levels Although many successful in vivo AMOstudies have been reported so far to our knowledge noneof them have used liposome-like delivery systems In ourprevious study we confirmed that YSK05-MENDefficiently releases siRNAs into the cytoplasm in a pH-dependent manner (30) It is with this property that webelieve we were able to achieve efficient AMO deliveryin miceTogether here we showed not only the potency of

20-OMe-40-thioribonucleosidendashmodified AMOs but alsothe utility of YSK05-MEND for AMO delivery Furtherin vivo studies leading to the understanding of the mech-anism of the 20-OMe-40-thioribonucleosidendashmodifiedAMO function as well as their potential side effect arecurrently under way

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online

ACKNOWLEDGEMENT

The authors would like to thank Ms Y Misawa(Hokkaido University) for technical assistance

FUNDING

Grant-in-Aid for Scientific Research [23249008] Grant-in-Aid for Scientific Research on Innovative ArealsquolsquoNanomedicine Molecular Sciencersquorsquo [2306] from theMinistry of Education Culture Sports Science andTechnology in Japan Funding for open access Grant-in-Aid for Scientific Research [23249008]

Conflict of interest statement None declared

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22 NakaT MinakawaN AbeH KagaD and MatsudaA (2000)The stereoselective synthesis of 40-b-thioribonucleosides via thePummerer reaction J Am Chem Soc 122 7233ndash7243

23 HoshikaS MinakawaN and MatsudaA (2004) Synthesis andphysical and physiological properties of 40-thioRNA applicationto post-modification of RNA aptamer toward NF-kB NucleicAcids Res 32 3815ndash3825

24 HoshikaS MinakawaN KamiyaH HarashimaH andMatsudaA (2005) RNA interference induced by siRNAsmodified with 40-thioribonucleosides in cultured mammalian cellsFEBS Lett 579 3115ndash3118

25 HoshikaS MinakawaN ShionoyaA ImadaK OgawaN andMatsudaA (2007) Study of modification patternndashRNAi activityrelationships by using siRNAs modified with 40-thioribonucleosides Chembiochem 8 2133ndash2138

26 KatoY MinakawaN KomatsuY KamiyaH OgawaNHarashimaH and MatsudaA (2005) New NTP analogs thesynthesis of 40-thioUTP and 40-thioCTP and their utility forSELEX Nucleic Acids Res 33 2942ndash2951

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29 TakahashiM NagaiC HatakeyamaH MinakawaNHarashimaH and MatsudaA (2012) Intracellular stability of20-OMe-40-thioribonucleoside modified siRNA leads to long-termRNAi effect Nucleic Acids Res 40 5787ndash5793

30 SatoY HatakeyamaH SakuraiY HyodoM AkitaH andHarashimaH (2012) A pH-sensitive cationic lipid facilitates thedelivery of liposomal siRNA and gene silencing activity in vitroand in vivo J Control Release 163 267ndash276

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miR-122 regulation of lipid metabolism revealed by in vivoantisense targeting Cell Metab 3 87ndash98

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40 DavisS ProppS FreierSM JonesLE SerraMJKinbergerG BhatB SwayzeEE BennettCF and EsauC(2009) Potent inhibition of microRNA in vivo withoutdegradation Nucleic Acids Res 37 70ndash77

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