Repeated Application of Sequence-Specific SiRNA Molecules Leads to an Effective Downmodulation of All Clinically Relevant bcr-abl Gene Variants.

Fusion transcripts such as bcr-abl encoding pathological oncogenic proteins represent ideal targets for a tumor-specific RNA interference (RNAi) approach. The aim of the present study was to optimize the efficacy of bcr-abl RNAi. We evaluated several synthetic siRNAs targeting the fusion sites of all common bcr-abl transcript variants (e14a2, e13a2, or e1a2). Significant knock-down of p210^Bcr-abl and p190^Bcr-abl fusion proteins was successfully achieved in bcr-abl expressing 32D cells and human leukemic K562 and MEG-01 cells. Repeated application of siRNA proved to be significantly more efficient compared to single treatment. The optimized protocol led to a total decrease of up to 75–90% in Bcr-abl protein levels, which was accompanied by a loss of viability of up to 90%. The target specificity of the siRNA was high, and even a single point mutation in the siRNA-sequence led to significant, albeit not complete, loss of siRNA efficacy. To expend the duration of the knock-down effect, we explored the use of plasmids driving the stable expression of short hairpin RNA (shRNA). Efficient downregulation of Bcr-abl protein was achieved in K562 cells transfected with a pSUPER-based plasmid encoding bcr-abl shRNA.