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

特洛伊木马：使用病毒样颗粒作为无脊椎动物体内的 RNA 传递装置作为害虫控制策略

• 批准号: BB/V009087/1
• 负责人: George Lomonossoff
• 金额: $ 70.56万
• 依托单位: John Innes Centre
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV009087%2F1
• 关键词: Trojan; Horse; Using; virus; like

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.

该提案旨在引发21世纪农业害虫防治的一场革命。自二十世纪中叶以来，害虫主要通过使用化学农药来控制，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，其使用越来越受到限制。然而，害虫仍然有可能对全世界种植的植物造成巨大损害，无论是通过取食造成的直接损害还是通过向植物传播疾病。因此，需要用更安全、更具体的“绿色”替代方法来取代化学害虫防治方法。该提案旨在通过向害虫提供专门设计的 RNA 分子来实现这一目标。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干扰的目标基因序列，或表达哪种蛋白质来产生毒性，可以确保该产品对它将影响的昆虫物种具有非常明确的针对性。该提案的真正新颖之处在于所提出的将这些生物活性 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 分子包装到蛋白质壳中，该蛋白质壳将在 RNA 传递给昆虫时对其进行保护。这些蛋白壳是植物病毒豇豆花叶病毒 (CPMV) 的病毒样颗粒 (VLP)：它们是非感染性纳米颗粒，很容易在植物中大量生产，并且由于最近的发现，可以用于专门包装任何选择的 RNA 分子。在这项工作过程中，我们将设计针对 JIC 昆虫研究所提供的五种害虫物种之一的特定活性的 RNA 分子。其中两种昆虫是飞蛾，两种是甲虫，一种是蚜虫，生物活性 RNA 的传递在某些昆虫物种中可能比其他昆虫物种更有效，因此使用一系列物种会增加我们成功的机会。我们将通过喂养实验来测试我们在这些昆虫中的生物活性 RNA 传递系统，在这些实验中我们测试了一系列定制的 RNA 分子。我们还将通过添加肽来对 CPMV VLP 的表面进行修饰，这些肽旨在提高对特定物种的特异性靶向性，或者提高 VLP 对昆虫细胞的渗透性。我们还将比较不同类型的 RNA 分子进行 RNA 干扰：我们可以采用不同的策略，它们在导致目标昆虫基因关闭方面可能具有不同程度的有效性。最后，最有前途的 VLP 和最有前途的定制 RNA 货物将在现实的害虫防治条件下进行测试，以确定其作为绿色农药的性能。目标昆虫种群将被允许在受控条件下以它们喜欢的寄主植物为食，并且将比较对照植物和喷洒了 VLP 农药的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、环境可持续的农业害虫防治方法，这将有利于所有利益相关者，从生物技术部门到农民和消费者。

项目成果

Specific Packaging of Custom RNA Molecules into Cowpea Mosaic Virus-like Particles.

将定制 RNA 分子特定包装成豇豆花叶病毒样颗粒。

• DOI: http://dx.10.1007/978-1-0716-2241-4_7
• 发表时间: 2022
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Peyret H

LoCKAmp: lab-on-PCB technology for

LoCKAmp：PCB 实验室技术，可实现不到 3 分钟的病毒基因检测。

• DOI: http://dx.10.1039/d3lc00441d
• 发表时间: 2023
• 期刊: Lab on a chip
• 影响因子: 6.1
• 作者: Papamatthaiou S