application): The goal of this pilot project is to explore the potential of RNA replication for prolonging the synthesis of messenger RNAs in the context of gene therapy for cystic fibrosis. RNA replication will be catalyzed by the RNA-dependent RNA polymerase that is encoded by one of the nodaviruses, a family of small, spherical, RNA viruses of insects, with bipartite single-stranded RNA genomes. The larger segment of the viral genome replicates autonomously when introduced into a wide variety of cells, and also supports the replication of other RNAs that carry appropriate cis-acting signals. In recent work, we used a hormone-inducible RNA polymerase II promoter to initiate vigorous RNA replication from the nuclei of cultured cells transfected with the RNA replicase cDNA. We have now designed self-contained DNA cassettes to launch replication of the mRNAs for several different reporter genes. The levels and duration of reporter gene expression will be compared with and without mRNA replication, in cultured cells and in the lungs of mice, thereby allowing us to determine the contribution of RNA replication to the abundance and longevity of gene expression in these two situations. Finally, we will attempt to achieve the replicative amplification of CFTR mRNA in this system and examine its expression in cultured cells, including primary airway cells from CF patients.
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