Fig 4. Inhibition of HCV IRES-mediated translation by systemically delivered LNP-formulated siIE22.

(A and B) Schematic diagram of siIE22 LNP (A) and LNP particle size analysis (B). (C) Experimental schedule and schematic representation of the pDual-IRES plasmid. The pDual-IRES plasmid was hydrodynamically injected through the tail vein of BALB/c mice (n = 4 per group). After 1 h, mice were iv injected with siIE22 LNP at a dose of 1 mg/kg body weight. The Fluc expression level in the liver was determined 16 h after the injection. Luciferase activity is reported as RLU per mg protein. *, P < 0.01. (D) BALB/c mice (n = 4 per group) were iv injected with indicated siRNAs (1 mg/kg body weight) complexed with ND98 or formulated with LNP. Poly(I:C) (1 mg/kg) complexed with ND98, formulated with LNP, or free form (each in 170 μl) was administered. PBS or LNP vesicles alone were used as control treatments. Two hours later, serum IFN-α levels were quantified by ELISA. The dotted line indicates the detection limit of the assay (15 pg/ml). (E) hPMBCs grown in 96-well plates were transfected with indicated siRNA at 10 nM concentration or with 1 μg/well poly(I:C) using the lipidoid ND98 or stimulated by a direct addition of 50 μg/ml poly(I:C) to the medium. After 16 h, cell culture supernatants from stimulated cells were analyzed for IFN-α by ELISA. Data shown are from one of the two independent experiments with similar results. ND, non-detectable. (F) HEK293 cells were transfected with the luciferase expressing plasmids (IFNβ-pGL3 and pRL-TK) for the IFN-β promoter activity assay. After 6 h, cells were transfected with 100 nM siIE22 or scrambled (Sc) siRNA, or 1 μg/ml poly(I:C). After 8 h, cells were harvested for dual luciferase assays. Fluc activity was normalized to Rluc activity from the pRL-TK plasmid. Normalized luciferase activity (Fluc/Rluc) of mock-treated cells was defined as 100. Data are presented as the mean ± SD of six measurements from two independent experiments.