Self-amplifying non-coding rna for targeted gene knockdown

Abstract

We describe the construction of a self-replicating form of microRNA (miRNA) and demonstrate its amplification and biological activity in cultured cells. In typical embodiments, a miRNA sequence of interest is flanked by self-cleaving ribozymes and added to RNAl of the Nodamura virus, which is directly translated to generate an RNA-dependent-RNA-polymerase that replicates it strongly along with the doubly-self-cleaving ribozyme cassette containing the miRNA. In the specific case of miR-34a flanked by Hammerhead and Tetrahymena ribozymes, BHK-21 cells transfected with the replicon form of this cassette are shown to generate up to one million copies/cell of the miRNA. The biological activity of this amplified miR-34a is demonstrated by the essentially complete apoptosis of cultured prostate cancer cells transfected with the replicon. More generally, we provide this self-amplifying form of miRNA as a means for restoring homeostatic levels of miRNA in cancer cells, as well as for targeting specific gene expression.

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