RNAi methods for Zika virus vector control

寨卡病毒载体控制的 RNAi 方法

• 批准号: 9325840
• 负责人: SUBBA R PALLI
• 金额: $ 21万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-14 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9325840
• 关键词: Adult; Aedes; Alpha Cell; Animals; Applied Research; Attenuated; Bacterial Toxins; Basic Science; Beds; Biochemistry; Biocompatible Materials; Biological Assay; Biology; Caenorhabditis elegans; Cell Line; Cells; Cellular biology; Cessation of life; Chemicals; Chemistry; Chitosan; Colorado; Confocal Microscopy; Culicidae; Dengue; Development; Diptera; Disease; Disease Vectors; Double-Stranded RNA; Drug or chemical Tissue Distribution; Effectiveness; Encephalitis; Filarial Elephantiases; Formulation; Gene Targeting; Genes; Goals; Health; Hemiptera; Histidine; Human; Inductively Coupled Plasma Mass Spectrometry; Insect Vectors; Insecta; Insecticides; Intracellular Transport; Knock-in; Label; Laboratories; Larva; Lepidoptera; Lysine; Malaria; Measurement; Methods; Modeling; Modernization; Molecular Biology; Nanostructures; Nanotechnology; Nematoda; Order Coleoptera; Particulate; Phase; Plants; Polymers; Potato; Powder dose form; Prevention; Principal Investigator; Problem Solving; Property; RNA Interference; RNA Processing; Research; Residual state; Resistance; Resistance development; Resolution; Small Interfering RNA; Spectroscopy, Fourier Transform Infrared; Technology; Time; Toxic effect; Transmission Electron Microscopy; Vector-transmitted infectious disease; Work; X ray diffraction analysis; X-Ray Diffraction; Yellow Fever; Zika Virus; analytical method; base; chikungunya; comparative; design; disorder control; experimental study; feeding; fighting; gel electrophoresis; improved; interdisciplinary approach; knock-down; larval control; light scattering; mortality; nano; nanocomposite; nanoformulation; nanostructured; novel; novel strategies; pathogen; programs; prototype; tool; uptake; vector; vector control

PROJECT SUMMARY Mosquitoes transmit zika virus as well as other pathogens that cause many deadly diseases. These vectors cause more human suffering than any other group of animals and are responsible for millions of deaths worldwide. Insecticide sprays, use of repellents and insecticide-treated bed nets for adult control and use of larvicides are the most common methods used to control disease vectors. However, widespread resistance development to insecticides in mosquitoes, human health and environmental concern from the residual toxicity of these chemicals are limiting the use of these strategies. Therefore, there is an urgent need to develop novel strategies to control disease vectors. Many applications of RNA interference (RNAi) are being developed for use in human health. RNAi could also help with studies in vector biology as well as in developing advanced vector control methods. RNAi efficiency is variable among insects; works very well in coleopteran insects but not in insects belonging to order Diptera that includes mosquitoes. One of the main challenges in the widespread use of RNAi technology in disease vectors is the lack of efficient and reliable dsRNA delivery method. We propose to develop novel formulations of double-stranded RNA (dsRNA) composited with polymers and/or particulate cores resulting in nanostructured delivery vehicles, or dsRNA nanocomposites (dsRNA-NCs). The major goal of these studies is to improve RNAi efficiency in mosquitoes that transmit Zika virus for applications in basic research as well as in the development of vector control methods. In specific Aim 1, we will prepare dsRNA-NCs and determine their properties. We hypothesize that biodegradable and environmentally friendly biomaterials could be used to develop prototype dsRNA-NCs that could aid in enhancing RNAi efficiency in mosquitoes. We will prepare cores of Ca3(PO4)2 coated with chitosan, Poly-L-Lysine or Poly- L-histidine. Additionally, Au cores will be used as an insoluble probe. A number of analytical methods will be used to determine the properties of synthesized dsRNA-NCs. We will also perform a comparative analysis of synthesized dsRNA- NCs by studying protection of dsRNA from dsRNases, its uptake, tissue distribution, intracellular transport including endosomal escape and dsRNA processing in Aeg-2 cells (a cell line developed from Aedes aegypti), Ae. aegypti larvae and adults as a model vector species. In Specific Aim 2, we will determine the efficacy and non-target effects of synthesized dsRNA-NCs in Ae. aegypti larvae and adults. We will expose Aeg-2 cells or feed Ae. aegypti larvae and adults with dsRNA- NCs and determine their efficiency in knocking down target genes and causing mortality as well as their effect on the expression of non-target genes. This application proposes to employ novel approaches, including nanotechnology to improve RNAi for use in disease vectors. The proposal uses interdisciplinary approaches to exploit modern technology for improvement in the health of citizens. Successful completion of proposed research will provide novel tools to study the biology of vectors of Zika and other pathogens and develop modern vector control methods.

项目摘要 蚊子传播寨卡病毒以及其他导致许多致命疾病的病原体。这些载体导致 比任何其他动物群体都更痛苦，并对全世界数百万人的死亡负责。杀虫剂 最常用的方法是喷洒、使用驱虫剂和驱虫蚊帐来控制成虫，以及使用杀幼虫剂 用于控制疾病媒介。然而，蚊子对杀虫剂的广泛耐药性发展，人类健康 以及对这些化学品残留毒性的环境关注限制了这些策略的使用。因此，我们认为， 迫切需要开发新的策略来控制病媒。RNA干扰（RNAi）的许多应用 正在被开发用于人类健康。RNAi还可以帮助载体生物学的研究以及开发 先进的病媒控制方法。RNAi效率在昆虫中是可变的;在鞘翅目昆虫中效果很好， 不存在于包括蚊子在内的双翅目昆虫中。广泛使用的主要挑战之一是， RNAi技术在疾病载体中缺乏高效可靠的dsRNA递送方法。我们建议发展 与聚合物和/或颗粒核复合的双链RNA（dsRNA）的新制剂， 纳米结构的递送载体或dsRNA纳米复合物（dsRNA-NC）。这些研究的主要目的是改善 RNAi在传播寨卡病毒的蚊子中的效率，用于基础研究以及 病媒控制方法。在具体目标1中，我们将制备dsRNA-NC并确定其性质。我们假设 可生物降解和环境友好的生物材料可用于开发原型dsRNA-NC， 增强RNAi在蚊子中的效率。我们将制备用壳聚糖、聚-L-赖氨酸或聚-L-赖氨酸包覆的Ca 3（PO 4）2核。 组氨酸此外，将使用Au芯作为不溶性探针。将采用多种分析方法， 确定合成的dsRNA-NC的性质。我们还将对合成的dsRNA进行比较分析- 通过研究dsRNA对dsRNases的保护，其摄取，组织分布，细胞内转运， Aeg-2细胞（从埃及伊蚊发育的细胞系）中的内体逃逸和dsRNA加工，Ae.埃及伊蚊幼虫和 成年人作为一种模式病媒物种。在具体目标2中，我们将确定合成的 在Ae.埃及伊蚊幼虫和成虫。我们将暴露Aeg-2细胞或喂养Ae。埃及伊蚊幼虫和成虫的dsRNA- NCs，并确定它们在敲除靶基因和引起死亡方面的效率以及它们对细胞的影响。 非靶基因的表达。本申请提出采用新的方法，包括纳米技术，以改善 用于疾病载体的RNAi。该提案采用跨学科方法，利用现代技术， 改善公民健康。成功完成拟议的研究将提供新的工具，研究 研究寨卡病毒和其他病原体的载体生物学，并开发现代载体控制方法。