Background

Our heavy reliance on chemical pesticides has resulted in increased incidences of insecticide resistance in pest insect populations and increasing negative impacts on non-target species. A new generation of species limited insecticides based on double-stranded RNA is being developed by an increasing number of research groups worldwide. Delivery of double-stranded RNA to an organism will result in sequence specific mRNA degradation in a process known as RNA interference (RNAi). By delivering a double-stranded RNAs that target gene(s) critical for growth or development of the insect, it is possible to kill the insect by selectively reducing the targeted gene’s expression.

Different insect species display differential responses to exogenously applied dsRNAs. Some insects including many beetles, are highly sensitive to dsRNAs, requiring only small quantities of dsRNAs to kill the insect. Other insects, in particular lepidopteran species (butterflies and moths), can be highly refractory to double-stranded RNA, and fail to show any RNAi-mediated transcript knockdown unless very high doses of dsRNAs are delivered. One factor that can strongly impact RNAi efficacy in an insect is the ability of the dsRNAs to enter targeted cells and to disperse throughout the cytoplasm to reach the targeted mRNA molecules. In some RNAi-refractory lepidopterans, the inability of the dsRNAs to reach the cytoplasm of the gut cells of the feeding insects is considered a primary reason why these insects fail to respond to ingested dsRNAs.

In most insect RNAi applications, dsRNA is administered to cells or insects as long (>200 bp) dsRNA. Short dsRNAs (<60 bp) are generally regarded as ineffective inducers of RNAi, as they have failed to be taken up by gut cells in various insects. However, short hairpin RNAs (shRNAs) have been used successfully in a limited number of insects. With these shRNAs, a single-stranded dsRNA is folded over on itself to create a short (typically 21-25 bp) double-stranded RNA structure with a single-stranded closed-loop. These shRNAs are generally considered more stable, and potentially more resistant to denaturation then the conventional linear dsRNA molecules.

Technology Overview

Invention Summary

Manitoba researchers have developed a synthetic RNA molecule that, following synthesis, folds to form a secondary structure known as a “paperclip” comprising:

a) a double stranded region comprising an RNAi sequence of at least 21 nucleotides;

b) two single‑stranded hairpin loops flanking either end of the double‑stranded region; and

c) a 5’ end and a 3’ end within the double stranded region that are closed but not covalently sealed.

While conventional dsRNAs, with their two collinear RNA strands, enter insect cells by clathrin-mediated endocytosis, the paperclips RNAs can enter cells by a clathrin-independent mechanism. Although the molecular mechanism of this clathrin-independent uptake process has not been identified, the fact that the pcRNAs can enter insect cells by an alternative mechanism will prove useful in the delivery of dsRNAs to insects that have either developed uptake resistance or they are naturally recalcitrant to uptake of double-stranded RNA, including lepidopteran pests.

Stage of Development

TRL 4

Benefits

A dsRNA with a novel paperclip dsRNA (pcRNA) configuration, with two closed ends, but not covalently sealed is described. This dsRNA has been developed as an alternative to conventional (linear) dsRNAs for insecticidal applications. Like other dsRNAs, it can be designed to be species-specific, thereby providing pest insect control without adversely affecting non-target species.

PcRNAs enter insect cells by a clathrin-independent manner, unlike other conventional dsRNAs that use clathrin-mediated endocytosis as the main mode of cellular entry. PcRNAs can therefore provide an alternative dsRNA to overcome dsRNA uptake resistance, which has already been demonstrated in lab-selected insects.

PcRNAs enter lepidopteran (moth) cells and induce RNAi, unlike conventional dsRNAs, which generally fail to induce RNAi in this large group of ecumenically-important pests. Hence, these new molecules can be used to control pests that are currently refractory to dsRNA.

Applications

Delivery of insect RNAi applications.

Opportunity

The University is actively seeking a licensee to license the patent to move this technology to market. The University also welcomes interactions with any investor or entrepreneur that would like to license the patent to create a start‑up based in Manitoba, or outside of the province.

The University would also welcome a company to invest in this research in Sponsored Research to further identify how the pcRNAs enter insect cells by a clathrin-independent manner.

Paperclip RNA Structure to Inhibit Target Gene Expression

Patents

IP Status

Seeking