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

这项建议旨在引发一场世纪农业害虫控制的革命。自世纪中期以来，昆虫害虫主要通过使用化学杀虫剂来控制，但由于这些产品对环境以及包括人类在内的动物健康的影响，它们的使用日益受到限制。然而，昆虫害虫仍然有可能对世界各地种植的植物作物造成巨大的损害，无论是通过喂养造成的直接损害，还是通过将疾病传播给植物。因此，需要用更安全和更具体的“绿色”替代品取代化学虫害控制方法。该提案旨在通过将专门设计的RNA分子传递给昆虫害虫来实现这一目标。这些RNA分子将通过RNA干扰（关闭对昆虫生存至关重要的基因）或瞬时表达（导致昆虫表达有毒蛋白质）发挥作用。通过选择RNA干扰靶向的基因序列，或表达引起毒性的蛋白质，可以确保产品对它将影响的昆虫物种具有非常特异性。这项提议真正新颖的是将这些生物活性RNA分子传递给昆虫的方法：我们将使用一种新开发的技术将定制RNA分子包装到蛋白质外壳中，这种蛋白质外壳将在RNA传递给昆虫时保护RNA。这些蛋白质壳是植物病毒豇豆花叶病毒（CPMV）的病毒样颗粒（VLP）：它们是非感染性的纳米颗粒，很容易在植物中大量产生，并且由于最近的发现，可以特异性地包装任何选择的RNA分子。在这项工作的过程中，我们将设计RNA分子，使其在JIC昆虫实验室现有的五种害虫中具有特异性活性。这些昆虫物种中有两种是飞蛾，两种是甲虫，一种是蚜虫，并且很可能生物活性RNA的递送在某些昆虫物种中比其他昆虫更有效，因此使用一系列物种增加了我们成功的机会。我们将通过喂养实验在这些昆虫中测试我们的生物活性RNA递送系统，在这些实验中，我们测试了一系列定制的RNA分子。我们还将对CPMV VLP的表面进行修饰，通过添加旨在改善对特定物种的特异性靶向的肽，或者应该改善VLP渗透到昆虫细胞中的肽。我们还将比较用于RNA干扰的不同类型的RNA分子：我们可以采用不同的策略，它们在导致靶昆虫基因关闭方面可能具有不同的有效性水平。最后，将在现实的害虫控制条件下测试具有最有希望的定制RNA货物的最有希望的VLP，以确定它作为绿色农药的效果如何。将允许目标昆虫的种群在受控条件下以其优选的宿主植物为食，并且将比较对照植物和已经用VLP杀虫剂喷洒的那些植物之间的昆虫存活率。我们的长期目标是开发针对无脊椎动物的生物防治方法，以取代对化学农药的依赖。该项目的目标是开发一种新颖、安全、环境可持续的农业虫害防治方法，这将有利于从生物技术部门到农民和消费者的所有利益相关者。

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.