The Highly Conserved5’ Untranslated Region As An Effective Target Towards The Inhibition Of Enterovirus71Replication By Chemically Modified Sirnas

Background and Objectives：Enterovirus71(EV71) has been implicated in numerous epidemics of hand, foot, andmouth disease (HFMD), which primarily affects infants and young children. Significantmorbidities and neurological complications associated with increasingly frequent HFMDoutbreaks throughout the Asia-Pacific region have raised public concerns. Currently,EV71is recognized as the most clinically important neurotropic virus since poliovirus hasbeen nearly eradicated in the majority of countries around the world. Despite EV71hasbeen discovered more than40years, treatments for acute EV71infections are mainlyaimed towards alleviating clinical symptom, and there is still no specific antiviral agentavailable against EV71.RNA interference (RNAi) is a biological and specific post-transcriptional genesilencing mechanism that has been employed in the development of antiviral therapeutics. Epidemiological analysis revealed that EV71viruses circulating throughout China do nothave great genetic diversity at least in recent10years. Ascertaining conserved siRNAsthat inhibit the replication of multiple EV71China strains was therefore feasible. In bothcell cultures and animal experiments, RNAi in the form of siRNA or plasmid-derivedshRNA has been used to effectively inhibit EV71infection, revealing several potentialtarget sequences within the3’UTR, VP1,3D,3C, and2C regions of the EV71genome.However, no effective siRNAs targeting the conserved5’untranslated region (UTR) of theEV71genome for specific viral inhibition have been reported. The5’UTR of EV71isinvolved in key steps of the viral life cycle. Due to its high degree of conservation whichrenders it less likely to mutate and functional importance for viral replication, the5’UTRof the EV71genome is an attractive target region for siRNA therapeutic. Screening ofeffective siRNAs targeting the conserved5’UTR of the EV71genome might have broadantiviral effect on diverse EV71China strains. Recent studies have successfully improvedsiRNA performance by incorporation of chemically modified RNA analogues againstvirus infection. Identifying the appropriate chemical modifications will be essential tobringing RNAi to the clinic.The application of unmodified21nt siRNAs to target highly conserved5’UTR of theEV71genome for specific viral inhibition was investigated in this study. Identification ofeffective target sequences within the highly conserved5’UTR and optimization of siRNAproperties by chemical modification for future in vivo applications were consideredprimary objectives. Furthermore, we investigated whether these siRNAs have broadantiviral effect on diverse EV71China strains. And then, we evaluated the antiviralactivity of chemically modified siRNAs on EV71-infected mice. These strategies mayultimately lead to the development of appropriate chemically modified siRNAs againstclinical EV71infection.Methods：1. Unmodified21nt double-stranded siRNAs were designed to target the5’UTR ofthe EV71genome by the method of multiple feature integration.2. The potential cytotoxicity of unmodified siRNAs was detected by MTT assays. 3. Cell viability assay and real-time TaqMan RT-PCR assay were carried out toscreen effective siRNAs.4. Si-1and si-2which found to effectively inhibit EV71replication were chemicallymodified by2’-OMe or2’-F at U and C sequences on complementary strands.Unmodified and chemically modified siRNAs (si-1, si-1OMe, si-1F, si-2, si-2OMe, si-2F)were used in this study.5. Confocal laser scanning biological microscope and flow cytometry were used toconfirm the optimal transfection efficiency.6. Comparison of cytotoxic effects as well as RNAi activity, immune stimulationand serum stability of the unmodified siRNAs and chemically modified siRNAs.7. Analysis of nucleotide sequence variations corresponding to the siRNAs in EV71China strains availabled in Genbank in the previous5year.8. According to nucleotide sequence analysis, the study was carried out on anotherEV71China strains EU703812(The genome of EU703812is characterized bycompletely match with si-1corresponding115-133nt sequence, a single nucleotidemismatch with si-2corresponding648-666nt sequence and this same single nucleotidemismatch up to41.66%in EV71China strain genomes available in Genbank in theprevious5years). We compared the inhibitory effect of siRNAs on two EV71Chinastrains.9. The si-2had been changed a nucleotide to completely corresponding the genomeof EU703812, and modified. The inhibitory effect of adjusted unmodified and chemicallymodified siRNAs on strain EU703812was evaluated by cell viability assay, real-timeTaqMan RT-PCR assay and immunofluorescence assay.10. Through the experiment on one-day-old ICR sucking mice infected with EV71,we first explored the experimental conditions.11. The potential toxicity of chemically modified siRNAs was detected byobserving the symptom, weight and survival of sucking mice.12. The antiviral activity of chemically modified siRNAs was evaluated byobserving the symptom, weight and survival of infected mice. Furthermore, real-timeRT-PCR analysis, histopathology and immunohistology of small intestine tissues in infected sucking mice were carried out to evaluate the antiviral activity of chemicallymodified siRNAs.Results：1. Screening out effective siRNAs targeting the conserved5’UTR of the EV71genome(1) Unmodified siRNAs targeting the5’UTR of the EV71genome that satisfied thescreening criteria were designated as si-1, si-2, si-3, si-4, and si-5.(2) All the21nt siRNAs used in this study did not exhibit any cytotoxic effectswhich could affect the viability and growth of RD cells.(3) Transfection of RD cells with si-1or si-2targeting the EV71genome5’UTRsignificant increased cell viability and resulted in significant reduction in EV71RNAtranscripts. The inhibitory effect on EV71replication was sequence-specific.2. The highly conserved5’UTR as an effective target towards the inhibition ofEV71replication by unmodified and chemically modified siRNAs in vitro(1) At6h post-transfection, fluorescein labeled si-2FAM could be seen localized inthe cytoplasm of RD cells by confocal laser scanning biological microscope. Withincreasing si-2FAM concentration, a corresponding increase of si-2FAM amounts in RDcells was observed. Furthermore, flow cytometry provided precise measurements oftransfection efficiency by enumerating fluorescein-labeled RD cells. The percentages offluorescein-labeled cells treated with si-2FAM at25nM,50nM, and100nM were79.47±1.79%,87.73±2.67%,97.17±0.86%, respectively. Therefore, siRNA concentrations at50nM and100nM were determined to be sufficient and used for the following RNAiexperiment.(2) Cytotoxicity assay showed that all the siRNAs used in this study, regardless ofthe observed time point or chemical modification, did not exhibit any cytotoxic effectswhich could affect the viability and growth of RD cells.(3) Appropriate chemically modified siRNAs exhibited similar RNAi activity incomparison with unmodified counterparts. Transfection of RD cells with chemicallymodified siRNAs (si-1F, si-2OMe, si-2F) targeting the EV71genomic5’UTR significantly delayed and alleviated the cytopathic effects of EV71infection, increasedcell viability in EV71infected RD cells. The inhibitory effect on EV71replication wassequence-specific and dosage-dependent, with significant corresponding decreases in viralRNA, VP1protein and viral titer. The cells transfected with si-2OMe and si-2F targetingthe sequence of nt648-666within the5’UTR of the EV71genome had better RNAiactivity than si-1F targeting the sequence of nt115-133within the5’UTR of the EV71genome. The chemically modified si-1OMe showed significantly decrease in antiviralactivity compared with unmodified counterpart.(4) There was no increase in PKR expression in the RD cells transfected withunmodified and chemically modified siRNAs. These siRNAs did not elicit any interferonresponse and the inhibition of EV71replication is mediated through RNAi.(5) When incubated in10%FBS, undiluted mouse serum, or undiluted humanserum, the unmodified si-2was not detected after6h. Conversely, the chemicallymodified si-2OMe and si-2F showed striking stability and were detected for at minimumof48h under the same conditions.3. The inhibitory efficiency of siRNAs against EV71China strains(1) When compared with EV71China strain genomes available in Genbank in theprevious5years, siRNAs targeting the115-133nt sequence of the EV71genomic5’UTR indicated the rates of sequences with complete nucleotide coincidence reachedabout71.30%, while the siRNAs targeting the648-666nt sequence of the EV71genomic5’ UTR showed the rates of sequences with complete nucleotide coincidence (37.04%)and those with the same single-nucleotide difference (41.66%) together reached about79.70%.(2) The antiviral activity of si-1against HM003207and EU703812were equallyeffective. The antiviral activity of si-2against EU703812was lower than HM003207, butalso significantly increased cell viability and resulted in significant reduction in EV71RNA transcripts.(3) Further studies showed that transfection of RD cells with adjusted unmodifiedand chemically modified siRNAs targeting EU703812significantly increased cell viability, resulted in significant reduction in EV71RNA transcripts and EV71-specificproteins.4. Inhibition of EV71in virus-infected mice by chemically modified siRNAs(1) We initially established EV71-infected model on one-day-old ICR sucking mice.EV71-infected sucking mice can appear apathetic, reduced activity. The symptomsdisappeared at6～7days post-infection. The time of death was more in24～96h post-infection.30%of the infected sucking mice died. EV71-infected sucking mice had lowerweight than DMEM-inoculated sucking mice within11days post-infection. Weightchanges were most obviously at6～7days post-infection. The EV71RNA can bedetected in small intestine, lung and skeletal muscle of sucking mice at3,5daypost-infection. At7day post-infection, the EV71RNA can not be detected in skeletalmuscle. At14day post-infection, the EV71RNA can not be detected in all the tissues ofsucking mice. Histological change of small intestine tissues in EV71-infected mice wasobvious at5day post-infection. Small intestine tissues can appear intestine epithelial cellvacuolar degeneration.(2) The study on potential toxicity of chemically modified siRNAs showed thatsi-2A-OMe and si-2A-F did not exhibit any toxicity on sucking mice.(3) In vivo experiment had showed that chemically modified siRNAs targeting theEV71genomic5’UTR alleviated symptoms of infected sucking mice, improved thesurvival rate of infected sucking mice, alleviated histological changes in small intestineof infected sucking mice, resulted in significant reduction in EV71RNA transcript levelsand EV71-specific VP1proteins expressions in small intestine tissues of infected suckingmice.Conclusions：Two siRNAs targeting the conserved5’UTR of the EV71genome were found toeffectively inhibit EV71replication. The sequences of nt115-133and nt648-666wereidentified within the highly conserved5’UTR that can be targeted to effectively inhibitEV71replication through RNAi strategies. This is the first study on the chemicallymodified siRNAs against EV71infection. The appropriate chemically modified siRNAs were identified that can effectively inhibit EV71replication. Nucleotide sequenceanalysis and verification on different EV71China strain indicated siRNAs targeting the115-133nt and648-666nt sequence of the EV71genomic5’UTR may have broadantiviral effect on diverse EV71China strains. The siRNAs targeting the648-666ntsequence of the EV71genome had better antiviral activity. A mixture of the siRNAs(including consideration of the same single-nucleotide mismatch of target sequences todesign a pair of siRNAs) targeting these two regions above may have much broaderantiviral effect on diverse EV71China strains. Further in vivo evaluation explored theeffectiveness of chemically modified si-2A-OMe and si-2A-F in inhibiting viralreplication in EV71-infected mice. These results provide a promising gene therapyapproach for EV71infection and also represent an important step in advancing siRNA tothe clinic in the future.