Breakpoint-Specific Targeting of p210^BCR-ABL mRNA by siRNA Alters Progenitor Cell Proliferative Rate in CML.

Abrogating expression of p210^BCR-ABL or of associated cell-signalling proteins in CML through siRNA-targeted destruction of mRNA has enormous potential in CML, both as a research tool and as a potential therapy, especially for imatinib resistant disease. We have investigated a p210^BCR-ABL junction-specific (b3a2) siRNA. We used an Oligofectamine transfection protocol which gives high transfection rates with minimal toxicity. In the Bcr-Abl-positive cell line, K562, this siRNA reduced p210^BCR-ABL expression by up to 80% compared with actin controls and induced significant levels of apoptosis. Silencing of p210^BCR-ABL has been reported by Scherr et al (Blood 2003, 101:1566–69) not to induce apoptosis in primary Ph+ cells. Similarly, we showed that the initial CFU-GM plating efficiency from b3a2 CML peripheral bloods treated with breakpoint-specific siRNA was not significantly reduced (100+/−5% of untreated controls). Replating large numbers of individual CFU-GM and analysing the distribution of secondary colonies can quantify the proliferative capacity of the myeloid progenitor pool. Using this assay, we showed that siRNA transfection significantly reduced the proliferative capacity of CML CFU-GM in a breakpoint-specific fashion (56+/−18% of untreated controls). This reduction in proliferative capacity is very similar to the CML-specific reduction in proliferative capacity induced by Interferon-alpha (50+/−40% of untreated controls) or by imatinib mesylate (50+/−30% of untreated controls). To our knowledge, this is the first demonstration of a functional consequence of siRNA treatment of primary CML haemopoietic cells. Our findings support the view that siRNA-based therapies may ultimately be of benefit, particularly in the management of CML