Measles virus (MV), a member of the Genus Morbil-livirus, the Family Paramyxoviridae, is an enveloped,negative-stranded RNA virus (14). The non-segmentedMV genome is composed of six genes that encodenucleocapsid (N), phospho (P), matrix (M), fusion (F),hemagglutinin (H) and large (L) proteins. The P geneharbors two additional overlapping accessory genes thatencode the C and the V proteins. The L protein isknown to be the principal catalytic component of theviral RNA-dependent RNA polymerase that is essentialfor replication of MV.

Subacute sclerosing panencephalitis (SSPE) is a fataldisease caused by mutant MV after prolonged infectionin the central nervous system. It has been reported thatabout 10 to 20 out of a million MV-infected individualsbecome SSPE (24). A combination therapy using inter-feron and inosiplex (1, 12) or ribavirin (16) has beenapplied to treat SSPE patients and contributed to extendtheir survival time to a certain level. However, com-plete cure of SSPE has not yet been achieved and newtherapeutic strategy is urgently needed.

RNA interference (RNAi) is the post-transcriptionalgene silencing caused by small RNA molecules that tar-get mRNA in a sequence-specific manner. In brief,double-stranded RNA is cleaved by Dicer, an RNaseIII-like enzyme, to generate short interfering RNA(siRNA) of 21–23 nucleotides. siRNA forms a com-plex, called RNA-induced silencing complex (RISC),and binds to its target mRNA in a sequence-specificmanner. It was first discovered in Caenorhabditis ele-gans (11) and, subsequently, in plants and insects (4,10, 15, 40). This phenomenon has also been observedin mammalian cells (9, 13, 23, 27, 28, 30, 39). siRNAswere reported to inhibit replication of a wide variety ofviruses, including human immunodeficiency virus (6,18), hepatitis C virus (35), and parainfluenza virus (3).Also, we previously reported that siRNAs that targetMV L mRNA, either chemically synthesized or plas-mid-based, efficiently inhibited the replication of MVK52 strain or SSPE-Kobe-1 strain (26).

Generation of Recombinant Adenovirus ExpressingsiRNA against the L mRNA of Measles Virus andSubacute Sclerosing Panencephalitis Virus

Momoko Otaki1, Da-Peng Jiang1, Mikiko Sasayama1, Motoko Nagano-Fujii1, and Hak Hotta*, 1, 2

1Division of Microbiology, and 2International Center for Medical Research and Treatment, Kobe University Graduate Schoolof Medicine, Kobe, Hyogo 650–0017, Japan

Received April 14, 2007; in revised form, July 11, 2007. Accepted July 20, 2007

Abstract: Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by pro-longed persistent infection of the central nervous system with a measles virus (MV) mutant called SSPEvirus. At present, there is no effective treatment to completely cure SSPE and development of a new ther-apeutic measure(s) against this fatal slow virus infection is needed. We previously reported that replicationof MV and SSPE virus was effectively inhibited by small interfering RNA (siRNA), either chemically syn-thetic or plasmid-driven ones, that were targeted against different sequences of the mRNA for the L pro-tein of MV. In this study, we have generated recombinant adenovirus expressing the siRNAs (rAd-siRNA-MV-L2, -L4 and -L5) and demonstrated that these rAd-siRNAs efficiently inhibited replication of MV andSSPE virus in a dose-dependent manner. Due to their high capacity for gene delivery to nerve cells and thepotential to inhibit SSPE virus replication, the rAd-siRNAs could be a good candidate for a novel thera-peutic measure against SSPE.

Key words: siRNA, Recombinant adenovirus, Subacute sclerosing panencephalitis, Measles virus

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Abbreviations: DMEM, Dulbecco’s modified Eagle medium;m.o.i., multiplicity of infection; MV, measles virus; PFU, plaque-forming unit; rAd-siRNA, recombinant adenovirus expressingsiRNA; RNAi, RNA interference; siRNA, small interferingRNA; SSPE, subacute sclerosing panencephalitis; TCID, tissueculture-infecting dose.

*Address correspondence to Dr. Hak Hotta, Division ofMicrobiology, Kobe University Graduate School of Medicine,7–5–1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650–0017, Japan.Fax: �81–78–382–5519. E-mail: hotta@kobe-u.ac.jp

At present, there are two major siRNA delivery sys-tems: one utilizing viral vectors such as adenovirus (2,19), adeno-associated virus (8, 37) and lentivirus (21,29), and the other utilizing non-viral vectors such aslipoplex (33) and polyplex (36). The adenoviral vectorpossesses a wide range of cell tropism, including non-dividing cells like a nerve cell, and, once incorporatedinto the cells, is able to express the gene of interest for along period of time considerably. In this study, we con-structed recombinant adenoviruses expressing siRNAsagainst the L mRNA of MV and examined their efficacyto inhibit the replication of SSPE virus.

Materials and Methods

Cells and MV/SSPE virus strains. Vero/SLAM cells(25) were a kind gift from Dr. Y. Yanagi, Department ofVirology, Kyushu University Faculty of Medical Sci-ences, Fukuoka, Japan, and were maintained in Dulbec-co’s modified Eagle medium (DMEM) supplementedwith 10% fetal bovine serum and G418 (400 µg/ml).293T cells, derived from human embryonic kidney,were also maintained in DMEM containing 10% fetalbovine serum.

A clinical isolate of MV (K52 strain) (20) and arecently isolated SSPE virus (SSPE-Kobe-1 strain) (17)were described elsewhere. They belong to genotype D3of MV. The infectivity of cell-free and cell-associatedviruses were determined based on syncytium formationand expressed as plaque-forming units (PFU)/ml.

Recombinant adenovirus expressing siRNAs (rAd-siRNA). rAd-siRNA was generated by AdMaxTM Ade-novirus Vector Creation system (Microbix Biosystems,Inc., Toronto, Ontario, Canada), according to the manu-facturer’s protocol. In brief, expression cassette ofsiRNA, which was designed to target the L mRNA ofthe MV genome, was derived from the pcPUR�U6iplasmid-based MV siRNA (26) and cloned into theBamHI and BglII multi-cloning site of pDC312 shuttleplasmid (Microbix Biosystems, Inc.). The shuttle plas-

mid construct was cotransfected with pBHGlox∆E1,3Cre harboring the entire adenoviral genome (serotype5) into 293T cells by using FuGene 6 reagent (Roche).After 15–18 days of cultivation, virus was extractedfrom the infected cells by freezing and thawing sixtimes. The titers of rAd-siRNA in the supernatant weredetermined and expressed as 50% tissue culture-infect-ing dose (TCID50).

Antiviral assay of rAd-siRNA. Vero/SLAM cellswere first inoculated with rAd-siRNA at multiplicities ofinfection (m.o.i.) of 30 to 3,000 TCID50/cell for 1 hr at37 C, and then infected with MV at m.o.i. of 0.03PFU/cell. After 2 days of cultivation, the supernatantswere collected and MV infectivity was measured onVero/SLAM cells. In another series of experiments, thecells were first infected with MV and then inoculatedwith rAd-siRNA.

Assessment of inhibitory effects of rAd-siRNA onSSPE-Kobe-1 virus replication was performed asreported previously with some modifications (26). Inbrief, 6 hr after cell seeding, SSPE virus-infected oruninfected Vero/SLAM cells were inoculated with rAd-siRNA at indicated m.o.i. and cultivated overnight.The following day, the SSPE virus-infected, rAd-siRNA-inoculated cells were cocultured with uninfectedcells and the number of syncytia that emerged on themonolayer cells was counted after 2 days. In anotherseries of experiments, SSPE virus-infected cells werecocultured with uninfected cells, and, 6 hr later, wereinoculated with rAd-siRNA for 1 hr. The number ofsyncytia was counted after 2 days of cultivation.

Results

Generation of rAd-siRNAWe previously reported that siRNAs targeted against

the L mRNA of the MV genome (MV-L2, -L4 and -L5)efficiently inhibited replication of both MV and SSPEvirus (26). We generated three strains of rAd-siRNAeach expressing either one of the three different siR-

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Table 1. The target sequences of siRNA and the titers of rAd-siRNA

siRNA Positiona) Sequence Titer (TCID50/ml)

MV-L2 141 to 161 5'-gaa cau caa gca ccg ccu aaa-3' 2.9�108

(9374 to 9394)MV-L4 311 to 331 5'-gga aga ucc gug agc ucc uaa-3' 1.7�108

(9544 to 9564)MV-L5 429 to 449 5'-gga cau caa gga gaa aau uau-3' 3.3�108

(9662 to 9682)

a) Nucleotide positions in the viral L mRNA. Parentheses indicate the correspondingpositions on cDNA to the genomic RNA of the Ichinose-B95a strain of MV (EMBL/Gen-Bank/DDBJ accession number AB016162).

NAs. The target sequences of the siRNAs and the titersof the siRNA-expressing rAd-siRNA are summarized inTable 1.

Inhibitory Effects of rAd-siRNA on MV ReplicationWe examined the possible inhibitory effects of rAd-

siRNA expressing MV siRNAs (MV-L2, -L4 and -L5)on MV replication. As a control, we used recombinantadenovirus expressing NS3-2 siRNA, which wasdesigned to target the NS3 region of the hepatitis C virusgenome (35). In the first series of experiments, rAd-siRNA was inoculated to Vero/SLAM cells prior to the

987ADENOVIRUS EXPRESSING siRNA AGAINST SSPE VIRUS

Fig. 1. Inhibition of MV replication by rAd-siRNA. (A) Vero/SLAM cells were inoculated with rAd-siRNA express-ing siRNAs (MV-L2, -L4 and -L5) at m.o.i. of 30, 300 and 3,000 TCID50/cell. rAd-siRNA expressing irrelevant NS3-2 siRNA (35) served as a control (Cont. siRNA). Cultures without rAd-siRNA inoculation (Untreated) served asanother control. One, 6 and 24 hr after rAd-siRNA inoculation, the cells were infected with MV (K52 strain). MVtiters in the culture supernatants were determined 2 days after infection. Data represent the mean � SEM obtainedfrom three independent experiments, which are expressed as the percentages of the titer in the control wells (Cont.siRNA). *, P�0.001; †, P�0.01, compared with the control. (B) Cells were first infected with MV and, after an indi-cated time (1, 6 and 12 hr), were inoculated with each of the rAd-siRNAs at m.o.i. of 30, 300 and 3,000 TCID50/cell.MV titers were determined 2 days after infection. Data represent the mean � SEM obtained from three independentexperiments. †, P�0.01; §, P�0.05, compared with the control.

MV infection (Fig. 1A). Regardless of the timing (1, 6and 24 hr) before MV inoculation and siRNA sequences(MV-L2, -L4 and -L5), rAd-siRNA suppressed MVreplication in a dose-dependent manner, with almostcomplete inhibition being observed at m.o.i. of 3,000TCID50/cell. A tendency was noted that rAd-siRNAinoculation 6 and 24 hr before MV infection exertedstronger inhibitory effects than that 1 hr before MVinfection.

In the second series of experiments, rAd-siRNA wasinoculated to the cells after the MV infection (Fig. 1B).When inoculated 1 hr after MV infection, rAd-siRNAmarkedly inhibited MV replication in the cells. Signifi-cant inhibition in a dose-dependent manner was stillobserved when rAd-siRNA was inoculated to the cells 6hr after the MV infection. However, MV inhibitionbecame much weaker when rAd-siRNA was inoculated12 hr after MV infection.

No significant difference in MV inhibition wasobserved among the three rAd-siRNAs tested (rAd-siRNA-MV-L2, -L4 and -L5).

Inhibitory Effects of rAd-siRNA on SSPE Virus Replica-tion

Next, we examined the possible inhibitory effects ofrAd-siRNA on SSPE virus replication. SSPE virus-infected and uninfected cells were inoculated withsiRNA-expressing rAd-siRNA or control rAd-siRNA,and maintained overnight. The SSPE virus-infected,rAd-siRNA-inoculated cells were then trypsinized andcocultured with rAd-siRNA-inoculated uninfected cells.The number of syncytia that emerged on the monolayercells was counted after 2 days. The results obtainedclearly demonstrated that rAd-siRNA expressing MVsiRNAs (MV-L2, -L4 and -L5) significantly inhibitedsyncytia formation, suggesting that all of the three rAd-siRNA strains effectively inhibited SSPE virus replica-tion (Fig. 2A). The inhibitory effects of rAd-siRNAbecame weaker but were still observed when rAd-siRNA was inoculated to the cells 6 hr after SSPE virusde novo replication started (Fig. 2B).

Discussion

At present, there is no satisfactory treatment to cureSSPE and the RNAi strategy is among the potentiallypromising candidates of therapeutic measures againstthis fatal slow virus infection. We previously reportedthat synthetic and plasmid-driven siRNA against the LmRNA of the MV genome efficiently inhibited MV andSSPE virus replication in a cell culture system (26).The L mRNA encodes the L protein of MV, which is anRNA-dependent RNA polymerase essential for viral

988 M. OTAKI ET AL

Fig. 2. Inhibition of SSPE virus replication by rAd-siRNA. (A)Vero/SLAM cells persistently infected with SSPE-Kobe-1 andfresh uninfected cells were each inoculated with rAd-siRNAexpressing siRNAs (MV-L2, -L4 and -L5) at m.o.i. of 30, 300and 3,000 TCID50/cell. Cells inoculated with rAd-siRNAexpressing irrelevant NS3-2 siRNA (35) served as a control(Cont. siRNA), and those without rAd-siRNA inoculation anoth-er control (Untreated). After being cultivated overnight, the rAd-siRNA-inoculated, SSPE virus-infected cells were trypsinizedand cocultured with the rAd-siRNA-inoculated uninfected cells.Syncytia that appeared on the monolayer cells after 2 days werecounted. Data represent the mean � SEM obtained from threeindependent experiments, which are expressed as the percent-ages of the number of syncytium seen in the wells inoculatedwith Cont. siRNA. *, P�0.01, compared with the control. (B)Vero/SLAM cells persistently infected with SSPE-Kobe-1 anduninfected cells were trypsinized and cocultured. Six hours aftercell seeding, the cells were inoculated with each of the rAd-siR-NAs at m.o.i. of 30, 300 and 3,000 TCID50/cell. After 2 days,syncytia that appeared on the monolayer cells were counted.Data represent the mean � SEM obtained from three indepen-dent experiments, which are expressed as the percentages of thenumber of syncytium seen in the wells inoculated with Cont.siRNA. *, P�0.01, compared with the control.

RNA replication and is least abundant among all theMV proteins expressed (3, 14). Therefore, the LmRNA would be a good target for the RNAi strategy toefficiently inhibit replication of MV and SSPE virus.

The specificity of the rAd-siRNAs expressing MVsiRNAs (MV-L2, -L4 and -L5) has been assured by thefollowing observations: (i) The control rAd-siRNAexpressing an irrelevant siRNA that targets the NS3region of hepatitis C virus RNA (35) did not inhibitreplication of MV or SSPE virus (Figs. 1 and 2), sug-gesting that rAd-siRNA infection itself does not induceany non-specific antiviral response in the cells; (ii)Despite the efficient inhibitory effects on MV K52strain that has exactly the same sequence as MV-L2, -L4and -L5, those MV siRNA did not inhibit replication ofencephalomyocarditis virus (26), suggesting that theseMV siRNAs do not induce any antiviral response,including interferon, as the virus is known to be highlysensitive to interferon treatment (32); (iii) MV-L4 and -L5, whose target sequences differ by only a singlenucleotide (MV-L5) or two (MV-L4) from that ofEdmonston strain of MV, did not inhibit replication ofthe Edmonston strain (26), confirming a sequence-spe-cific inhibition mediated by MV siRNAs.

Compared with plasmid-based MV siRNAs, the rAd-siRNAs appear to need a longer time to fully expressinhibitory effects on MV replication. The rationale forthis statement is that rAd-siRNAs expressing MV siRNAs only partially inhibited MV replication wheninoculated to the cells 6 hr or 12 hr after MV inoculation(Fig. 1B) whereas plasmid-based MV siRNAs almostcompletely inhibited MV replication when used underthe same timings (26). These results, however, do notnecessarily disvalue the use of rAd-siRNAs as it canachieve efficient introduction of siRNA-expressing cas-settes into the target cells without using transfectionreagents, which may be potentially harmful in vivo.

Once SSPE virus replication has reached a certainlevel, the inhibitory effects of rAd-siRNAs were negatedto some extent (Fig. 2B), compared to the situation inwhich rAd-siRNAs were used before de novo replica-tion of SSPE virus started (Fig. 2A). In patients withSSPE, there are already many virus-infected cells andrAd-siRNAs expressing MV siRNAs may not com-pletely inhibit SSPE virus replication in the cells. How-ever, these siRNAs can confer resistance to the virusinfection in the surrounding uninfected cells, therebypreventing the spread of SSPE virus in the brain.

A number of important issues should be addressedwhen considering the possible clinical application ofsiRNA. Firstly, a good delivery system by which toefficiently introduce siRNAs into the brain cells is need-ed. Adenovirus virus vectors are known to achieve

high titers and, more importantly, transduce the gene ofinterest into non-dividing cells, including nerve cells(31, 34). In this study, therefore, we have generatedrAd-siRNA expressing siRNAs against the MVgenome, and demonstrated that they could mediate effi-cient inhibition of MV and SSPE virus replication(Figs. 1 and 2). Secondly, adverse effects of the genedelivery system should be avoided. In this connection,adenovirus vectors are less likely to mediate oncogenictransformation, compared to retrovirus and lentivirusvectors (31). Thirdly, we need to cope with viralescape from siRNA due to a mutation(s) in the targetsequences (5, 7, 13, 22, 38). SSPE virus frequentlyundergoes genetic mutations during its long-term per-sistence in a patient (14). To overcome this problem, wehave generated three strains of rAd-siRNA that targetdifferent sequences of the viral genome. As the nextstep towards the clinical application, the effectivenessof the rAd-siRNAs when applied in vivo and the safetywhen applied to the central nervous system should beevaluated carefully.

The authors are grateful to Dr. K. Taira, Graduate School ofEngineering, the University of Tokyo, Tokyo, Japan, and Dr. Y.Yanagi, Department of Virology, Kyushu University GraduateSchool of Medicine, Fukuoka, Japan, for providingpcPUR�U6i and Vero/SLAM cells, respectively. This workwas supported in part by Special Research Program for PrionDisease and Slow Virus Infection from the Ministry of Health,Labour and Welfare, Japan, and was also carried out as part ofthe 21COE Program at Kobe University Graduate School ofMedicine.

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