NUCLEIC ACID DECOYS TARGETING RNA PROTEIN DETERMINANTS OF MRNA STABILITY

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Gene expression is a logical and increasingly feasible target for therapeutic efforts. The identification of novel molecular targets for new classes of drugs, specifically in the area of neucleic acid based therapeutics, will be based on advances in the detailed understanding of genetic mechanisms and controls. Contributions of posttranscriptional controls to gene expression are increasingly evident. MRNA stability is of particular importance for mRNAs with unusually short (oncogenes, cell growth factors) or long (structural proteins) half-lives. Mechanisms by which mRNA stability is controlled have in common a central role for interaction of sequences in the mRNA 3'-untranslated region (UTR) with cytosolic protein factors to form sequence-specific ribonucleoprotein (RNP) complexes. Alteration in the structure and/or assembly of these complexes may allow a predictable and potentially therapeutic increase or decrease of gene expression. Such approaches will utilize novel molecular targets and mechanisms of action for nucleic acid based therapies. Globin mRNA, a well documented protype of a long-lived mRNA and one of the few mRNAs for which the determinants of RNA stability have been studied in detail, will serve as the model system for this proposal. Alpha-globin mRNA stability is dependent on the interaction of defined sequences in the 3'-UTR with a set of cytosolic RNA binding proteins. We hypothesize that inhibition of this mRNA/protein interaction will result in selective mRNA destabilization. This hypothesis will be tested in an integrated set of in vitro, cell based, and transgenic studies to establish the efficacy, mechanisms, and feasability of this approach. A series of decoy nucleic acids will be tested in vitro and in intact cells in culture for their effects on 3'-UTR RNP complex formation and for corresponding effects on mRNA stability. Potential decoys will include polyC homopolymers, multimers of the alpha-globin stability motif (CCUCC), and segments of the alpha-globin mRNA 3'UTR. Variables of chemistry, structure, and delivery systems will be assessed for their effects on efficiency and selectivity of decoy action. Mechanisms and specificity of observed activities will be investigated. Transgenic mouse lines will be established expressing optimized decoy RNAs under the control of conditional promoters to test their efficacy and characterize their function(s) in vivo. This approach, once validated, optimized, and mechanistically defined in the model system, can be adapted to target a broad spectrum of gene systems including those which control cell transformation and oncogenesis.