The eventual goal is to develop therapeutic agents capable of inducing cell suicide in specific types of cancer cells, by the regulated expression of a gene encoding the A-fragment of diphtheria toxin (DT-A). In principle, toxin gene expression could be directed to any cell type by harnessing regulatory elements form genes expressed preferentially in those cells, and by applying means to prevent expression in other cell types. In conjunction with efficient (probably viral) delivery vehicles, such genetically controlled toxic agents would offer the potential for the selective killing of cancer cells without toxic side effects in normal tissues. This proposal deals with the development of model systems for achieving stringent control over the expression of a toxin gene when introduced into mammalian cells in culture, using electroporation (specific aims (1) to (3)), or in transgenic mice as an in vivo model (aim #(4)). The primary focus will be on targeting B-lymphoid cells and subsets of B- cells for ablation by regulated expression of the DT-A coding sequence. Secondarily, the targeting of T-cells will be investigated as a potential system allowing inducible expression of DT-A, dependent on T-cell activation.
The specific aims are: 1) To make further improvements in DT- A expression constructs with specificity for B-lymphoid cells using regulatory elements from immunoglobulin genes, 2) To target DT-A expression to subsets of B-lymphoid cells using lg light chain or MHC class II gene regulatory elements, 3) To target DT-A expression to activated T-lymphoid cells using regulatory elements from the IL2 gene and to demonstrate cell suicide induced by T-cell activation, 4) To generate transgenic mice using several of the above constructs so as to enable rigorous assessment of tissue-specificity of DT-A expression in an in vivo model. These goals build on and extend the work performed during the initial grant period and their successful accomplishment will further progress towards the therapeutic application of regulated toxin gene expression. The ability to target B-cells or activated T-cells for ablation should have direct relevance for therapy of certain malignancies. Experience gained in use of characterized lymphoid-specific regulatory elements for targeting DT-A expression will also be valuable in adapting additional regulatory systems from more """"""""cancer-specific"""""""" genes for this purpose.
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