Table 1.
Small interfering RNA sequence information.
Table 2.
Bio-analytical assay primers.
Figure 1.
Small interfering RNA suppression of STAT6 expression in vitro.
A549 lung epithelial cells were transfected with a range of siRNA concentrations (0.1 – 100 nM) and total RNA and protein extracted 72 hours later. The percent STAT6 mRNA remaining at each of the concentrations tested was used to calculate the 50% inhibitory concentration of 372u (a) and 372 m (b); 0.27nM and 0.35nM, respectively. Western blot analysis was then used to confirm STAT6 protein ablation, which was observed at all concentrations of 372u tested (c) and at concentrations ≥ 5 nM for 372 m (d). SC = Silencer Select Negative Control No. 1 siRNA, vehicle = transfection reagent only. Values presented are mean ± S.E.M (n = 3).
Figure 2.
Optimisation of the bio-analytical assay.
(a-d): Maintenance of bio-analytical assay sensitivity within different biological matrices. 372u or 372 m siRNA were spiked into 1 × TE buffer (•), plasma (□) or rat lung tissue extract (▵) at a range of concentrations (0.032 – 100 pg/µl) and then analysed using an RT-PCR-based bio-analytical assay. No significant difference in the threshold cycle of detection (Ct) was noted between the three matrices at any of the siRNA concentrations of tested. Shown are representative standard curves obtained for sense (a, b) and anti-sense (c, d) strand measurement. Values presented are mean (n = 3) with errors bars omitted for clarity. Percent PCR efficiency for 372u (sense) in TE buffer, plasma, or lung extract = 94.2%, 86.3% and 102.8% respectively. Percent PCR efficiency for 372 m (sense) = 124.2%, 101.5%, 114.8% respectively. (e, f): To compare the effect of RNA extraction methods on bio-analytical assay sensitivity, rat lung tissue was spiked with 372u (e) or 372m (f) (125 ng) and homogenised in 4M guanidine isothiocyanate (GITC) or Tri-reagent. Tri-reagent homogenised tissue was processed through the manufacturer’s recommended procedure for the extraction of RNA, either up to the chloroform phase separation step (TR-AQ) or through the entire process (TR-F). Recovery of siRNA from TR-AQ processed tissue was comparable to GITC, recovery from TR-F processed tissue was significantly lower. Values presented are mean ± S.E.M (n = 3), and represent % siRNA recovered in comparison to GITC.
Figure 3.
In vitro Caco-2 cell monolayer toxicity testing of STAT6 siRNA.
Caco-2 cell monolayers were exposed to a range of siRNA concentrations (50 – 5000pM) for 6 hours and membrane integrity monitored by the transfer of Lucifer Yellow dye between the basolateral and apical compartments. No significant transfer of Lucifer Yellow dye was noted following treatment with either 372u (a) or 372m (b) compared to the vehicle control (saline). Analysis of siRNA recovered from the apical (c, d) and basolateral (e, f) compartments following treatment, showed that both 372u (c, e) and 372m (d, f) were present at ≤ 4pg/µl, LLOQ = 0.01 pg/µl). Values presented are mean ± S.E.M (n = 3), and P-values represent comparison to the vehicle (saline) control.
Figure 4.
Bio-distribution and persistence of STAT6 targeting siRNA in rat lungs following intra-tracheal delivery.
Normal rats were administered 372u or 372m (1 mg/kg, intra-tracheal) and siRNA quantification performed in lung tissue and plasma. Whole lung analysis showed the presence of significantly more 372m after 6 hours compared to 372u (b). 372u (c) and 372m (d) were both distributed throughout the lung 5 minutes after intra-tracheal administration. In addition, plasma concentrations of 372m (f) were significantly higher and persisted for longer than 372u (e). 1 = right accessory lobe, 2 = right cranial lobe, 3 = right middle lobe, 4 = upper left lobe, 5 = mid left lobe, 6 = lower left lobe, 7 = upper right caudal lobe, 8 = mid right caudal lobe and 9 = lower right caudal lobe. Values presented are mean ± S.E.M (n = 3), and P-value represents comparison between 372m and 372u.
Figure 5.
Effectiveness of STAT6 targeting siRNA within a rat model of allergic inflammation.
Ovalbumin-sensitised animals were administered 372u, 372m, MMC siRNA (2 mg/kg, intra-tracheal) or dexamethasone (0.3 mg/kg) on 3 consecutive days and then aerosol challenged with ovalbumin 24 hours after the final dose. Seventy-two hours after challenge, 372m was present at significantly higher concentrations in BAL than 372u (a, b). Treatment with 372u or 372m did not reduce lung inflammation compared to the saline treated control, as evidenced by histological scoring (c) and inflammatory cell enumeration (d-f). Treatment with dexamethasone however, did significantly reduce lung inflammation following allergen challenge. Values presented are mean ± S.E.M (n = 10), and P-value represents comparison to the saline treated control.
Figure 6.
Bio-ditribution of STAT6 targeting siRNA following intranasal delivery.
Fine dissection (f) followed by mRNA and protein analysis, revealed that STAT6 expression within the nasal cavity of Brown Norway rats was invariant (a, b). Anaesthetised animals were administered 2mg/kg of siRNA in sterile saline and bioanalytical analysis performed 72 hours later on fine-dissected tissue samples and plasma. Small interfering RNA was detectable throughout the nasal cavities (<0.1% original dose) with 372m present at significantly higher (P<0.001) levels in the SL compared to 372u (c, d). 372m persisted longer and was detected at significantly higher levels in rat plasma compared to 372u (e). Values presented are mean ± S.E.M (n = 6), and P-value represents comparison to 372u. Nasal sections = Lateral lining, LL; inferior ethmoid turbinate, IE; median ethmoid turbinate, ME; superior ethmoid turbinate, SE; maxilla-turbinate, M; naso-turbinate, N & septum lining, SL.