A key obstacle in the effective treatment of patients with cancer is to distinguish between tumour cells and normal cells, and this represents the major limitation for most of the current treatments. Through the de novo design of zinc finger proteins which can bind to any specific DNA sequences this distinction can be made by designing these to bind specifically to the genetic modifications which can be defined at the DNA sequence level in some cancers. This was recently demonstrated by developing a zinc finger protein which specifically recognized the unique DNA sequence at the breakpoints junction of the chromosomal translocation which creates the BCR-ABLp190 oncogene. The DNA binding of these proteins was shown to be effective in repressing transcription in vivo resulting in apopotosis of the cells. To realistically conceive of using this approach in patients we need first to be able to test its efficacy in an animal model system which recapitulates the DNA rearrangement events resulting in oncogene activation. We therefore plan to generate in the mouse the equivalent DNA rearrangement found in the human acute lymphoblastic leukaemia associated with the Philadelphia chromosome, which involves fusion of the BCR and c-ABL genes by translocation. To model this abnormality in-vivo we will develop a binary transgenic system to allow activation of a site-specific DNA recombination event that will be restricted to the lymphoid lineage. In this scheme, gene targeting in ES cells will be used to construct murine bcr and c-abl sequences containing loxP sites, which are short sequences which can be cut and rejoined by a recombinase Cre. The precise arrangement of the bcr and c-abl sequences will be such that in mice derived from these ES cells these will be in a non-functional configuration (i.e. they will not represent the structure of a murine equivalent of the BCR-ABLp190 oncogene) but when crossed with a strain expressing the Cre recombinase, site-specific recombination will occur in the progeny and result in construction of a bcr-ablp190 oncogene. The frequency and timing of the rearrangement will be controlled by the nature of the transcriptional control sequences driving expression of the Cre recombinase. Retroviral vectors encoding zinc finger peptide domains specific to the sequences involved in the chromosomal rearrangement event resulting in the bcr-ablp190 oncogene activation will be used to infect haematopoietic stem cells derived from the binary transgenic cross. Reconstitution of lethally irradiated recipients with these infected cells will then allow the therapeutic potential of the zinc finger peptides to be evaluated.
Funding SchemeCSC - Cost-sharing contracts
EH9 3JQ Edinburgh