Trojan Horse: Using virus-like particles as RNA delivery devices in invertebrates as a pest-control strategy

This proposal aims to spark a revolution in agricultural pest control for the 21st century. Since the middle of the twentieth century, insect pests have been controlled primarily through the use of chemical pesticides, but the use of these products is increasingly restricted because of their impact on the environment as well as animal health, including humans. However, insect pests still have the potential to cause huge amounts of damage to plant crops grown worldwide, either through direct damage caused by feeding or through the transmission of diseases to the plants. There is therefore a need to replace chemical pest control methods with safer and more specific "green" alternatives. This proposal aims to achieve this by delivering specially-designed RNA molecules to insect pests. These RNA molecules will act either through RNA interference (shutting off a gene that is critical to the insect's surival) or transient expression (causing the insect to express a protein that will be toxic). By choosing which gene sequence to target by RNA interference, or which protein to express to cause toxicity, it is possible to ensure that the product is very specific in which insect species it will affect. What is really novel about this proposal is the method that is proposed to deliver these bioactive RNA molecules to the insects: we will use a newly developed technique for packaging custom RNA molecules into protein shells that will protect the RNA as it is delivered to the insect. These protein shells are virus-like particles (VLPs) of the plant virus cowpea mosaic virus (CPMV): they are non-infectious nanoparticles that are easy to produce in large amounts in plants, and, thanks to recent discoveries, can be made to specifically package any RNA molecule of choice.
During the course of this work, we will design RNA molecules for specific activity in one of five insect pest species available at the JIC Insectary. Two of these insect species are moths, two are beetles, and one is an aphid, and it is likely that delivery of bioactive RNA will work better in some insect species than others, so using a range of species increases our chance of success. We will test our bioactive RNA delivery system in these insects through feeding experiments in which we test a range of custom RNA molecules. We will also make modifications to the surface of the CPMV VLPs, by adding peptides that are intended to improve specific targeting to a particular species, or that should improve penetration of the VLPs into the insect cells. We will also compare different types of RNA molecules for RNA interference: there are different strategies that we could adopt and they may have different levels of effectiveness at causing the target insect gene to be switched off. Finally, the most promising VLP with the most promising custom RNA cargo will be tested under realistic pest control conditions to determine how good it is at being a green pesticide. Populations of the target insect will be allowed to feed on their preferred host plant in controlled conditions, and insect survival will be compared between control plants and those that have been sprayed with the VLP pesticide. Our long-term aim is to develop invertebrate-specific biocontrols to replace the reliance on chemical pesticides.

The development of a novel, safe, environmentally sustainable approach to pest control in agriculture, the aim of the project, would be beneficial to all stakeholders, from the biotechnology sector to farmers and consumers.

This proposal aims to explore a novel method for sustainable pest control. Thanks to our improved understanding of RNA packaging in the plant-infecting cowpea mosaic virus (CPMV), we have developed a means of specifically packaging any RNA sequence of choice inside CPMV virus-like particles (VLPs): non-infectious protein shells that protect the custom RNA. These custom RNA - packaging VLPs are produced through transient expression in the plant Nicotiana benthamiana, from which they can be extracted and purified easily using either bench-scale protocols (regularly used at JIC) or industrial-scale manufacturing methods. During the course of this project, we aim to package bioactive RNAs into CPMV VLPs that will trigger either RNA interference or transient protein expression in our selected insect pest species (coleopteran, lepidopteran and hemipteran). Preliminary experiments carried out at JIC show that long hairpin dsRNA mimics are efficiently packaged into CPMV VLPs using our custom RNA packaging system, and we have already published that custom protein-coding RNA can be packaged in the VLPs. Moreover, preliminary experiments in vitro and in vivo indicate that VLPs can be taken up by insect cells and the RNA packaged within them can be translated. We have therefore devised a programme of work in which bioactive RNAs will be designed to either silence an insect gene or express a toxic protein in one of the target insect species. Thanks to the JIC Insectary facility, we will then feed VLPs containing these bioactive RNAs to the insects using artificial feeding protocols, and use standard techniques to determine RNA interference (RT-qPCR) or transient protein expression (western blotting). We will also modify the surface of the CPMV VLPs with various peptides intended to improve targeting specificity and efficiency. Finally, we will analyse the stability and effectiveness of the most promising VLP-RNA combination in realistic pest-control conditions.