(683e) Engineering Cancer-Specific Cis-Acting Intronic Regulators of Alternative Splicing as Novel Tools for Disease Detection and Treatment

The proper regulation of gene expression is critical to normal cellular function. Cells achieve precise spatial and temporal control over gene expression by coupling post-transcriptional regulation pathways, which is critical for complex cellular function such as differentiation and development. Alternative splicing is a process by which multiple protein isoforms are generated from a single coding region by altering the ways in which exons are joined together during the splicing process. This pathway is used by cellular systems to both increase proteomic diversity from a limited number of genes and to precisely control gene expression. Regulators of alternative splicing include cis-acting sequences within a transcript that control alternative splicing events. These elements play a key role in regulating the form and function of a protein isoform produced from a given gene in response to various signals received by the cell. Therefore, the potential exists to engineer ligand-controlled cis-regulators of alternative splicing to achieve precise temporal and spatial control over gene expression in mammalian cells. The development of platforms for the construction of such riboregulator elements will provide a flexible, modular technology for the targeted regulation of gene expression events, which will have significant application in the detection, treatment, and monitoring of various diseases such as cancer.

We have developed a novel in vivo selection strategy for generating cell-specific intronic regulators of alternative splicing events. This selection strategy has been used to screen a library of random 15 nucleotide inserts for sequences that exhibit functional activity as cancer cell-specific intronic splicing silencers and enhancers (ISSs and ISEs) within intronic regions of model fusion mini-gene constructs. The selection and characterization of cancer cell–specific regulators of alternative splicing will be discussed. Multiple rounds of selection and counter-selection between two prostate cancer cell lines that vary in their degree of metastasis were applied to an intronic library to generate prostate cancer cell-specific regulators of alternative splicing patterns. Those sequences exhibiting prostate cancer-specific splicing patterns were further characterized for consensus sequence motifs, functional modularity, and relative strengths as splicing regulators. The flexibility of this selection scheme to be generally applied to different cell lines will enable this strategy to be used for generating diverse groups of cell- and tissue-specific regulators of gene expression. Furthermore, the directed construction of cis-acting RNA switches that act as ligand-controlled regulators of alternative splicing patterns will be discussed. The application of this technology to early disease detection and the design of ‘intelligent', or targeted, therapeutics, where the disease treatment may be localized to a particular cell or tissue type or activated by specific cellular responses, will be discussed.