Although the World Health Organization has declared the eradication of smallpox in 1980, the fear of its potential use in bioterrorism has become a reality. Since the effectiveness of current vaccines and antiviral drugs is limited, development of new therapeutic strategies is needed. In this study, we investigated small interfering RNA (siRNA) as a therapeutic approach for preventing and treating smallpox infection.
Methods
Eight siRNA sequences were designed and evaluated for antiviral activity against vaccinia virus (VACV) in vitro and in vivo.
Results
Of eight siRNAs, A5R1 siRNA targeted the A5R gene and reduced VACV replication in cell culture by up to 85% at 100 nM concentration without inducing cytotoxicity. A prolonged prophylactic as well as therapeutic effect of siRNA was observed. In addition, real-time PCR analysis showed that A5R1 siRNA can especially reduce the target mRNA. Finally, intraperitoneal delivery of A5R1 siRNA in Balb/c mice significantly protected these animals from lethal challenge with VACV.
Conclusions
This study suggests the potential of A5R1 siRNA as a therapeutic antiviral agent against smallpox.
KennedyR.B., OvsyannikovaI., PolandG.F.Smallpox vaccines for biodefense.Vaccine2009; 27: D73–D79.
3.
ThèvesC., BiaginiP., CrubezyE.The rediscovery of smallpox.Clin Microbiol Infect2014; 20: 210–218.
4.
ParanN., SutterG.Smallpox vaccines: new formulations and revised strategies for vaccination.Hum Vaccin2009; 5: 824–831.
5.
GrosenbachD.W., JordanR., HrubyD.E.Development of the small-molecule antiviral ST-246 as a smallpox therapeutic.Future Virol2011; 6: 653–671.
6.
MuckerE.M., GoffA.J., ShamblinJ.D.Efficacy of Tecovirrmat (ST-246) in nonhuman primates infected with variola virus (smallpox).Antimicrob Agents Chemother2013; 57: 6246–6253.
7.
PecotC.V., CalinG.A., ColemanR.L., Lopez-BeresteinG., SoodA.K.RNA interference in clinic: challenges and future directions.Nat Rev Cancer2011; 11: 59–67.
8.
de FougerollesA., VornlocherH.P., MaraganoreJ., LibermanJ.Interfering with disease: a progress report on siRNA-based therapeutics.Nat Rev Drug Discov2007; 6: 443–453.
9.
GrimmD.Small silencing RNAs: state-of-the-art.Adv Drug Deliv Rev2009; 61: 672–703.
10.
NekhaiS., JerebtsovaM.Therapies for HIV with RNAi.Curr Opin Mol Ther2006; 8: 52–61.
11.
WuJ., NandamuriK.M.Inhibition of hepatitis viral replication by siRNA.Expert Opin Biol Ther2004; 4: 1649–1659.
12.
ButzK., RistrianiT., HengstermannA., DenkC., ScheffnerM., Hoppe-SeylerF.siRNA targeting of the viral E6 oncogene efficiently kills human papillomavirus-positive cancer cells.Oncogene2003; 22: 5938–5945.
13.
TompkinsS.M., LoC.Y., TumpeyT.M., EpsteinS.L.Protection against lethal influenza virus challenge by RNA interference in vivo.Proc Natl Acad Sci U S A2004; 101: 8682–8686.
14.
BurnettJ.C., RossiJ.J., TiemannK.Current progress of siRNA/shRNA therapeutics in clinical trials.Biotechnol J2011; 6: 1130–1146.
15.
HarrisonS.C., AlbertsB., EhrenfeldE.Discovery of antivirals against smallpox.Proc Natl Acad Sci U S A2004; 101: 11178–11192.
VigneS., GermiR., DuraffourS.Specific inhibition of orthopoxvirus replication by a small interfering RNA targeting the D5R gene.Antivir Ther2008; 13: 357–368.
19.
VigneS., DuraffourS., AndreiG., SnockR., GarinD., CranceJ.M.Inhibition of vaccinia virus replication by two small interfering RNA targeting B1R and G7L genes and their synergistic combination with cidofovir.Antimicrob Agents Chemother2009; 53: 2579–2588.
20.
ChouW., NgoT., GershonP.D.An overview of the vaccinia virus infection: a survey of the proteins of the poxvirus-infected cell.J Virol2012; 86: 1487–1499.
21.
BroylesS.S.Vaccinia virus transcription.J Gen Virol2003; 84: 2293–2303.
22.
AhnB.Y., RoselJ., ColeN.B., MossB.Identification and expression of rpo19 a vaccinia virus gene encoding a 19-kilodalton DNA-dependent RNA polymerase subunit.J Virol1992; 66: 971–982.
23.
ByrdC.M., BolkenT.C., HurbyD.E.The vaccinia virus I7L gene product is the core protein proteinase.J Virol2002; 76: 8973–8976.
24.
ByrdC.M., HrubyD.E.A conditional-lethal vaccinia virus mutant demonstrates that the I7L gene product is required for virion morphogenesis.Virol J2005; 2: 4.
LiB.J., TangQ., ChengD.Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque.Nat Med2005; 11: 944–951.
27.
KimD.H., RossiJ.J.Strategies for silencing human disease using RNA interference.Nat Rev Genet2007; 8: 173–184.
28.
SongE., LeeS.K., DykxhoornD.M.Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages.J Virol2003; 77: 7174–7181.
WangJ., LuZ., WientjesM.G., AuJ.L.S.Delivery of siRNA therapeutics: barrier and carrier.AAPS J2010; 12: 492–503.
31.
LarsonS.D., JacksonL.N., ChenL.A., RychahouP.G., EversB.M.Effectiveness of siRNA uptake in target tissues by various delivery methods.Surgery2007; 142: 262–269.
32.
CannJ.A., JahrlingP.B., HensleyL.E., Wahl-JensenV.Comparative pathology of smallpox and monkeypox in man and macaques.J Comp Pathol2013; 148: 6–21.
33.
LeconetW., PetitP., Peraldi-RouxS., BressonD.Nonviral delivery of small interfering RNA into pancreas-associated immune cells prevents autoimmune diabetes.Mol Ther2012; 20: 2315–2325.
