Use of lipid nanoparticles encapsulating nucleic acids for use in basic research on fish health and for the development of aquaculture therapeutics

封装核酸的脂质纳米粒子在鱼类健康基础研究和水产养殖疗法开发中的用途

• 批准号: RGPIN-2022-05070
• 负责人: Lee, Justin
• 金额: $ 1.93万
• 依托单位: University of the Fraser Valley
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=745779
• 关键词: Use; lipid; nanoparticles; encapsulating; nucleic

This research program is aimed at formulating, characterizing and developing lipid nanoparticle (LNP) systems encapsulating nucleic acids such as messenger RNA (mRNA) and small-interfering RNA (siRNA) for basic research on fish health and for the development of future aquaculture therapeutics. My specific focus is designing a treatment for viral hemorrhagic septicemia virus (VHSV). As LNP systems have never been researched on fish cells before, my research program will involve an intensive investigation using in vitro fish cell culture. LNPs encapsulating nucleic acids are an exciting and current therapeutic option. In August 2021, the FDA approved the LNP encapsulating mRNA (LNP-mRNA) therapeutic known as the Pfizer-BioNTech COVID-19 Vaccine (Comirnaty). Additionally, in December 2020 the FDA issued emergency use authorization for the Moderna COVID-19 vaccine (SPIKEVAX). Both Comirnaty and SPIKEVAX vaccines have been administered to billions of people globally in a concerted effort to end the COVID-19 pandemic.  Fluorescently-labeled LNPs encapsulating siRNAs will be used to examine cellular uptake in a variety of fish cells including rainbow trout (RT) gill epithelial cells (RTGillW1), intestinal epithelial cells (RTGutGC), macrophages (RTS11), olfactory and muscle cells. These fluorescently-labeled LNPs will also be used to examine the mechanism of uptake (endocytosis) into fish cells. Molecular inhibitors and fluorescent uptake markers will be used to determine the exact mechanism of uptake. Gene silencing capabilities will then be assessed by encapsulating GFP-siRNA in LNPs.  The LNP-GFP-siRNAs will then be examined for gene silencing capabilities in the RTS11 cell line stably expressing TurboGFP. In addition, other siRNA targets will be examined in the RTGillW1, RTGutGC, olfactory and muscle cell lines.  A major aim of this research is to design a novel therapeutic for VHSV, a disease which is known to cause up to 50% mortality (in rainbow trout) when introduced into bodies of water.  Several vaccines have previously been developed for VHSV including a plasmid DNA vaccine (that encodes the Glycoprotein (G) gene and a plasmid DNA that encodes an shRNA that induces sequence specific silencing of the (G) gene in VHSV-challenged cells. Both plasmid DNA therapeutics have shown success, but none have been approved as a commercial vaccine/treatment. This is because many nations do not approve these DNA vaccines as the rainbow trout treated with these vaccines would be labeled as genetically modified organisms (GMOs). Therefore, designing an LNP-siRNA system encapsulating G-siRNA directed against the G-gene is an excellent way to circumvent this problem. LNP-G-siRNA will be tested on VHSV-challenged RTGillW1 cells to test for sequence specific knockdown and to assess protection against VHSV. The ultimate goal is to be able to thoroughly research an effective LNP-G-siRNA VHSV treatment that can be commercially available globally.

该研究计划旨在制定，表征和开发脂质纳米颗粒（LNP）系统，该系统包含核酸（例如Messenger RNA（mRNA））和小型互换RNA（siRNA），以进行鱼类健康和开发未来水产养殖治疗的基础研究。我的具体重点是设计用于病毒出血性败血病毒（VHSV）的治疗方法。由于以前从未对LNP系统进行研究，因此我的研究计划将涉及使用体外鱼类细胞培养的大量投资。封装核酸的LNP是一种令人兴奋且目前的治疗选择。 2021年8月，FDA批准了封装MRNA（LNP-MRNA）疗法的LNP，称为Pfizer-Biontech Covid-19疫苗（Comirnaty）。此外，2020年12月，FDA颁发了现代Covid-19疫苗（Spikevax）的紧急使用授权。 Comirnaty和Spikevax疫苗均已为全球数十亿人提供，以结束19日大流行的一致努力。包裹siRNA的荧光标记的LNP将用于检查各种鱼类细胞中的细胞摄取，包括虹鳟（RT）g上皮细胞（RTGUTGC），巨噬细胞（RTS11），嗅觉和肌肉细胞。这些荧光标记的LNP也将用于检查摄取（内吞作用）对鱼类细胞的机理。分子抑制剂和荧光吸收标记将用于确定摄取的确切机制。然后，将通过将GFP-SIRNA封装在LNP中来评估基因沉默能力。然后，将检查LNP-GFP-SIRNA，以在RTS11细胞系中稳定表达涡轮增压FP中的基因沉默能力。此外，将在RTGILLW1，RTGUTGC，嗅觉和肌肉细胞系中检查其他siRNA靶标。这项研究的主要目的是设计一种针对VHSV的新疗法，VHSV疾病已知，当被引入水体时，该疾病会导致高达50％的死亡率（在彩虹鳟鱼中）。先前已经为VHSV开发了几种疫苗，包括质粒DNA疫苗（编码糖蛋白（G）基因和质粒DNA，该基因编码shRNA，该shRNA诱导了（g）基因的（g）基因在VHSV挑战细胞中的序列（g）均未得到验证，但许多人都没有批准。批准这些DNA疫苗作为用这些疫苗处理的彩虹鳟鱼将被标记为一般修饰的生物（GMO），因此设计了针对G-Gene的G-SiRNA的LNP-SIRNA系统，这是对LNP-G-Sirna的一个很好的测试。对VHSV的保护。