633. Dose-Dependent Gene Silencing by Lentivirus-Mediated RNA Interference (RNAi) in bcr-abl Positive Cells

Abstract

RNA interference (RNAi) describes a process of double-stranded RNA-dependent post-transcriptional gene silencing. This gene silencing is mediated by 21–23 nt small interfering RNAs (siRNAs) which induce sequence-specific mRNA degradation. Fusion-transcripts encoding oncogenic proteins may represent potential targets for a tumor-specific RNAi-approach. Recently, siRNAs have been shown to specifically inhibit expression of the bcr-abl oncogene essential for chronic myeloid and bcr-abl positive acute lymphoblastic leukemia (CML, ALL) in hematopoietic cell lines. Since RNAi triggered by a single siRNA application is only transient in mammalian cells potential anti-tumor effects are limited in the models studied so far. Gene transfer of transcription cassettes for suitable RNAi-triggers, however, can induce long-term gene silencing even in primary hematopoietic cells. Using lentiviral gene transfer to transcribe different shRNAs targeting the bcr-abl oncogene and to express red fluorescence protein (RFP) as a quantitative reporter to track cellular transduction we demonstrate that stable but not transient RNAi can efficiently deplete bcr-abl+human K562 and murine TonB cells. Importantly, killing of bcr-abl+cells depends on the dose of lentivirus used for transduction and correlates with the RFP-expression level of transduced cells. Furthermore, we show that lentiviral transduction of homogenous cell lines results in heterogenous cell populations in terms of lentiviral integrations, RFP-and siRNA expression, target gene silencing, and functional phenotype. Whereas transduced K562 cells with high RFP-expression are eradicated, a limited number of cells with lower RFP-expression and smaller number of lentiviral integrations may outgrow after longer periods of cell culture. Interestingly, these cells still show some reduction in bcr-abl mRNA levels, aberrant proliferation kinetics, and higher sensitivity to the Bcr-Abl kinase inhibitor STI571 as compared to controls. Furthermore, lentiviral gene transfer of anti bcr-abl shRNA into primary CML cells can inhibit colony formation of transduced CD34+progenitor cells by about 50%. These data demonstrate that lentivirus-mediated RNAi can induce high efficient and stable gene silencing depending on the number of lentiviral integrations in cell lines and in primary cells. Selection processes of gene modified cells, however, have implications for any potential therapeutic application and for functional genomic analysis based on gene transfer strategies to induce stable RNAi in mammalian cells.