But a recent study using siRNA against RSV in mouse lung showed a lack of detectable siRNA-induced interferon29

But a recent study using siRNA against RSV in mouse lung showed a lack of detectable siRNA-induced interferon29. TOR-2 used in the cell-culture study24, to strain PUMC01 used in the macaque model21,22 and to another 100 published SCV strains isolated during different phases of SCV evolution as recently defined6 with wide geographic distributions around the world; (ii) they are the two most potent inhibitors for reducing SCV replication in FRhK-4 cells among a set of active siRNA duplexes selected from 48 siRNA duplexes targeting the entire SCV genome23,24; (iii) a synergistic anti-SCV activity was observed when a combination of siSC2 Glycitein and siSC5 was applied in the cell-culture study showing the strongest prophylactic and therapeutic effects (Fig. 1b,c)24; (iv) their targeted sequences share no homology with the human genome, avoiding potential nonspecific knockdown of the endogenous genes of an individual receiving this type of treatment. Glycitein In addition, two unrelated siRNA duplexes, siCONa and siCONb, with no homology to either the human genome or the SARS genome, validated in the cell-culture study24, show no RNAi activity for SCV inhibition, and were chosen as the negative control (Supplementary Fig. 1 online). Open in a separate window Figure 1 Selection and validation of siRNA duplexes targeting SCV sequence.(a) The RT-PCRCamplified region is marked at the most upstream region of open reading frame 1 (ORF1). The siSC2- and siSC5-targeted regions (red dashes) were also marked within the Spike- and NSP12-coding regions of the SCV genome, respectively. Black arrows indicate the locations of the two targeted sequences within the viral RNA genome and gray arrowheads indicate mutation sites. Electron microscopy images of SCV particles are indicated by arrows within SCV-infected FRhK-4 cell (b) and the SCV-infected FRhK-4 cell treated with siSC2-5 (c). Scale bar in b and c, 200 nm. (d) Luciferase expression (measured in relative luciferase units, RLU) in mouse lungs after co-delivery of the expression plasmid pCI-scLuc and either siSC2-5 or siCONc-d, in either D5W or Infasurf solution. TIE1 * 0.05, = 5. siSC2 and siSC5 duplexes were active in mouse lung To Glycitein insure activity of the siSC2-siSC5 combination (siSC2-5) with a clinically viable delivery method we first established a luciferase-based reporter gene system, containing both siSC2 and siSC5 targeted sequences between a cytomegalovirus promoter and the luciferase coding region. Cotransfection of pCI-scLuc and siSC2-5 into cultured cells confirmed that siSC2-5 can specifically knock down luciferase expression (data not shown). To identify a clinically viable carrier for siRNA delivery into mouse lung, we selected two carriers currently in clinical use, D5W solution29 and Infasurf solution30, which have been applied in delivery of DNA31 and siRNA32 to animal models. Twenty-four hours after intratracheal administration of 30 g of pCI-scLuc plasmid DNA and 30 g of siSC2-5 in 100 l of D5W or Infasurf solution into BALB/c mouse lungs, we analyzed Glycitein luciferase expression in the lung tissues. Co-delivery of pCI-scLuc plasmid with siSC2-5 in D5W solution resulted in a higher level of reporter gene expression and a stronger RNAi effect than that delivered in the Infasurf solution (Fig. 1d). We noted that TransIT-TKO and polyethyleneimine have been reported as carriers for intranasal33, intratracheal34 and intravenous35 deliveries of siRNA into mouse models for treatment of influenza virus and respiratory syncytial virus infections. But those carriers are not feasible for clinical use,.

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