Stable RNA interference (RNAi) as an option for anti-bcr-abl therapy
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RNA interference (RNAi) has recently been used for sequence-specific gene silencing of disease-related genes including oncogenes in hematopoietic cells. To characterize its potential
therapeutic value, we analyzed different modes to activate RNAi as well as some pharmacokinetic aspects of gene silencing in bcr-abl+ cells. Using lentiviral gene transfer of transcription
cassettes for anti-bcr-abl shRNAs and red fluorescence protein (RFP) as a quantitative reporter, we demonstrate that stable but not transient RNAi can efficiently deplete bcr-abl+ K562 and
murine TonB cells from suspension cultures. Importantly, depletion of bcr-abl+ cells depends on the dose of lentivirus used for transduction and correlates with the RFP-expression level of
transduced target cells: RFP-high K562 cells are eradicated, whereas RFP-low or -intermediate cells may recover after prolonged cell culture. Interestingly, these cells still show reduced
bcr-abl mRNA levels, aberrant proliferation kinetics, and enhanced sensitivity to the Bcr-Abl-kinase inhibitor STI571. Quantitative PCR from genomic DNA suggests that more than three
lentiviral integrations are required for effective depletion of K562 cells. Finally, we demonstrate that lentivirus-mediated anti-bcr-abl RNAi can inhibit colony formation of primary CD34+
cells from chronic myeloid leukemia patients. These data demonstrate dose-dependent gene silencing by lentivirus-mediated RNAi in bcr-abl+ cells and suggest that stable RNAi may indeed be
therapeutically useful in primary hematopoietic cells.
This work was supported in part by grants of the HW and J Hector-Stiftung, the Wilhelm Sander-Stiftung, and the ‘Deutsche Forschungsgemeinschaft’ (SFB 566). We thank George Daley (MIT,
Cambridge) for providing us with the TonB cell line used in this study.
Department Hematology and Oncology, Hannover Medical School, Hannover, Germany
III Med. Clinic, University Hospital of Mannheim, University of Heidelberg, Mannheim, Germany
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