Functional RNAs: combined assembly and packaging in VLPs

Abstract

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.

Bioengineered Noncoding RNAs Selectively Change Cellular miRNome Profiles for Cancer Therapy

“…The recent termination of Phase I clinical study on synthetic miR-34a mimic (MRX34) (Beg et al, 2017), owing to high incidence of adverse immune responses, again testifies the body's capability to distinguish chemoengineered RNAi agents as foreign. Therefore, biologic approaches such as in vitro transcription (Beckert and Masquida, 2011) and, especially, bioengineering in live cells (Ponchon and Dardel, 2007;Ponchon et al, 2009;Huang et al, 2013;Li et al, 2014Li et al, , 2015Li et al, , 2018Chen et al, 2015;Wang et al, 2015;Pereira et al, 2016;Fang et al, 2017), are highly warranted to produce natural RNA molecules that should better represent cellular ncRNA properties for basic research and experimental therapy (Ho and Yu, 2016;Pereira et al, 2017).…”

RNA: packaged and protected by VLPs

“…13,14 The packaging of RNA by protein(s) in vivo, in coupled RNA production and packaging systems, may be effective for overcoming at least a subset of these challenges. 15 RNA expression in vivo [16][17][18] can employ recombinant nucleic acids that encode polymerase promoters, affinity tags, functional 'target' sequences and genes for packaging proteins.…”

“…[19][20][21] Qb VLPs are hollow with pores 1.4 nm in diameter. 20 Lau et al 22 and Fang et al 15 previously demonstrated packaging of non-viral RNA in Qb VLPs by co-expressing target RNA and CP from distinct plasmids in E. coli. In vivo packaging of target RNA in VLPs is promoted by an RNA hairpin (hp), derived from Qb RNA, which has high affinity for CP ( Fig.…”

Nanomaterials for Cancer Precision Medicine

“…As is well known, virus RNA carrier exhibits high transfection efficiency, but at the cost of strong immunogenicity. Virus‐like nanoparticles have therefore been developed as gene and drug carriers from different plant protein or Hepatitis B core protein …”