Functional RNAs: combined assembly and packaging in VLPs

We describe here a one pot RNA production, packaging and delivery system based on bacteriophage Qβ. We demonstrate a method for production of a novel RNAi scaffold, packaged within Qβ virus-like particles (VLPs). The RNAi scaffold is a general utility chimera that contains a functional RNA duplex with paired silencing and carrier sequences stabilized by a miR-30 stem-loop. The Qβ hairpin on the 5΄ end confers affinity for the Qβ coat protein (CP). Silencing sequences can include mature miRNAs and siRNAs, and can target essentially any desired mRNA. The VLP–RNAi assembles upon co-expression of CP and the RNAi scaffold in E. coli. The annealing of the scaffold to form functional RNAs is intramolecular and is therefore robust and concentration independent. We demonstrate dose- and time-dependent inhibition of GFP expression in human cells with VLP–RNAi. In addition, we target the 3΄UTR of oncogenic Ras mRNA and suppress Pan-Ras expression, which attenuates cell proliferation and promotes mortality of brain tumor cells. This combination of RNAi scaffold design with Qβ VLP packaging is demonstrated to be target-specific and efficient.