Killing a cell is a relatively straightforward affair, but selectively repairing damaged DNA is far more tricky.





Work Package 1: Design of the Gene Network

We are designing a vector system for rapid gene network construction and vector shuttling. The system will be flexible, allowing "modules" from the different project workpackages to be integrated. We are beginning with "Proof of principle" constructs; building constructs using components that are already available to us, such as oncogenic promoters, characterised meganucleases/zinc fingers, and other network proteins. In this way we will begin to build gene networks to detect and repair cancer mutations.

Work Package 2: Engineering of Cell lines with the modified pathways

The main goal is the generation of isogenic engineered cell lines where to test the viral gene network constructs to provide an homogeneous proof of principle for the activity of the constructs. These cell lines will have modifications in the essential molecules of the regulation of the p53 network. A second objective is to provide human tumour cell lines characterized for the alterations in the p53 network to test in complex systems the functionality of the viral constructs.

Work Package 3: New Meganucleases & Zinc Fingers

We will modify the specificity of meganucleases to recognize new target DNA sequences to enable targeted gene repair as a function of the sensor networks. Concurrently, we will engineer zinc fingers, attached to an endonuclease, to double-strand cleave selected DNA sequences and induce selective gene repair by homologous recombination. To facilitate the protein engineering task, we will be working on the FOLD-X algorithm to allow Computer-aided protein-DNA design.

Work Package 4: Delivery Systems

We will construct new conditionally replicating adenoviral vectors including complex p53 sensor networks for target cell discrimination. The vector properties, decisive for future therapeutic potential will be determined in a panel of artificial cell lines with defined alterations in the p53 pathway.

Work Package 5: Experimental evaluation of the designed Networks

This is the part of the work to which all the previous Work Packages (WP) contribute. Essentially it involves the testing of the designed gene networks, been delivered either through transformation or viral delivery. The WP includes the use of the different cell lines made in WP2, for which we control where the errors are, and in a final step the assay of our gene networks on tumour derived cell lines with aberrations on the p53/mdm2 pathway. Two types of assays will be carried out. On one hand we will deliver networks that will induce cell death when detecting an aberration. On the other systems that will induce correction of the altered genes. In the first case the reporter will be cell viability. In the second case, we will include a reporter that will express a GFP only when the pathway has been repaired. In both cases control cell lines with the same genetic background but no aberrations on the studied pathway will be used.