miRNA-Mediated Silencing of Modified mRNAs

Example 10

To test whether this miRNA dependent silencing of modified mRNAs is not limited to miR-126, GFP coding mRNA containing target sites for miR-21 or miR-145 was designed. Ad-HEH293 cells were transfected with miR-21, miR-145 or miR-143 mimics 24 hr prior to transfection with unmodified or pseudouridine and 5-methylcystidine modified GFP, GFP4x21TS or GFP4x145TS mRNAs to ensure for RISC assembly and the GFP expression levels were assessed after 24 hr. Cells transfected with 100% Pseudouridine and 5-methylcystidine substituted GFP4x21TS mRNA, the over-expression of miR-21 and not miR-145, miR-21 or moR-143 reduced the expression GFP to the same extent as cells transfected with unmodified GFP4x21TS mRNA (FIGS. 45A-45C). Cells transfected with 100% Pseudouridine and 5-methylcystidine substituted GFP4x154TS mRNA, showed the same reduction in GFP expression as unmodified mRNA when miR-145 mimic was added and not when miR-21, miR-126 or miR-143 mimics were added (FIGS. 45A-45C). In the control transfected cells with unmofdified or 100% substitution with Pseudouridine and 5-methylcystidine GFP mRNA (with no miRNA target sites), the over-expression of miR-21, miR-145 or miR-143 did not have an effect on GFP expression (FIGS. 45A-45C).