Functional VEGFA knockdown with artificial 3'-tailed mirtrons defined by 5' splice site and branch point.

Kian Hong Kock,Shawn Hoon,Kiat Whye Kong,Yiqi Seow

doi:10.1093/nar/gkv617

Abstract

Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs, and will be especially useful for co-delivery of coding genes and RNAi. A specific family of mirtrons – 3′-tailed mirtrons – has hairpins precisely defined on the 5′ end by the 5′ splice site and 3′ end by the branch point. Here, we present design principles for artificial 3′-tailed mirtrons and demonstrate, for the first time, efficient gene knockdown with tailed mirtrons within eGFP coding region. These artificial tailed mirtrons, unlike canonical mirtrons, have very few sequence design restrictions. Tailed mirtrons targeted against VEGFA mRNA, the misregulation of which is causative of several disorders including cancer, achieved significant levels of gene knockdown. Tailed mirtron-mediated knockdown was further shown to be splicing-dependent, and at least as effective as equivalent artificial intronic miRNAs, with the added advantage of very defined cleavage sites for generation of mature miRNA guide strands. Further development and exploitation of this unique mirtron biogenesis pathway for therapeutic RNAi coupled into protein-expressing genes can potentially enable the development of precisely controlled combinatorial gene therapy.

Highlights

The therapeutic potential of the RNA interference (RNAi) pathway, in which short double-stranded RNA mediate translational repression or degradation of targeted mRNAs, has been explored extensively with exogenous mimics such as small interfering RNAs [1], short-hairpin RNAs [2,3] or artificial miRNAs [4]
We demonstrate synthetic tailed mirtrons (TMirts) designs capable of mediating slicing-dependent gene knockdown for a variety of targets, most notably reducing VEGF protein levels in a hypoxia-induced model
We further show that TMirts can be more efficacious in gene knockdown, compared to miRNA mimics and canonical mirtrons that utilize the same guide strand

Summary

Introduction

The therapeutic potential of the RNA interference (RNAi) pathway, in which short double-stranded RNA mediate translational repression or degradation of targeted mRNAs, has been explored extensively with exogenous mimics such as small interfering RNAs (siRNAs) [1], short-hairpin RNAs (shRNAs) [2,3] or artificial miRNAs (amiRNAs) [4]. Canonical mirtrons suffer from sequence constraints due to splicing requirements that limit targetable sequences. There is a subclass of mirtrons with a 3 tail that is remarkable because most of the sequences required for splicing, the polypyrimidine tract and the branch point, are located outside of the hairpin (Figure 1a) [9], freeing up sequence constraints (purine-rich for 5 arm guide strands; pyrimidine-rich for 3 arm) for synthetic mirtron design. For natural 3 -tailed mirtrons (TMirts), the precise biogenesis pathway from spliced intron to a hairpin substrate for exportin-5 and Dicer has not been elucidated, but has previously been shown to require further nucleolytic processing in flies [9]. Successful development of artificial TMirts capable of targeting therapeutic genes of interest will have benefits for combinatorial gene therapy approaches

Methods

Results

Conclusion