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，该系统封装了信使RNA （mRNA）和小干扰RNA （siRNA）等核酸，用于鱼类健康的基础研究和未来水产养殖治疗方法的开发。我的研究重点是设计一种治疗病毒性出血性败血症病毒（VHSV）的方法。由于LNP系统从未在鱼细胞上进行过研究，因此我的研究计划将涉及对鱼细胞体外培养的深入研究。包裹核酸的LNPs是一种令人兴奋的当前治疗选择。2021年8月，FDA批准了LNP封装mRNA （LNP-mRNA）疗法，即辉瑞- biontech COVID-19疫苗（Comirnaty）。此外，FDA于2020年12月发布了Moderna COVID-19疫苗（SPIKEVAX）的紧急使用授权。在共同努力结束COVID-19大流行的过程中，全球数十亿人接种了Comirnaty和SPIKEVAX疫苗。荧光标记的LNPs包封sirna将用于检测各种鱼类细胞的细胞摄取，包括虹鳟鱼（RT）鳃上皮细胞（RTGillW1）、肠上皮细胞（RTGutGC）、巨噬细胞（RTS11）、嗅觉和肌肉细胞。这些荧光标记的LNPs也将用于研究摄取（内吞作用）进入鱼类细胞的机制。分子抑制剂和荧光摄取标记将用于确定摄取的确切机制。然后通过在LNPs中封装GFP-siRNA来评估基因沉默能力。然后在稳定表达TurboGFP的RTS11细胞系中检测lnp - gfp - sirna的基因沉默能力。此外，将在RTGillW1、RTGutGC、嗅觉和肌肉细胞系中检测其他siRNA靶点。这项研究的一个主要目的是为VHSV设计一种新的治疗方法，这种疾病被引入水体后（在虹鳟鱼中）可导致高达50%的死亡率。以前已经开发了几种针对VHSV的疫苗，包括编码糖蛋白(G)基因的质粒DNA疫苗和编码shRNA的质粒DNA，该shRNA可诱导VHSV挑战细胞中(G)基因的序列特异性沉默。两种质粒DNA疗法都显示出了成功，但没有一种被批准作为商业疫苗/治疗方法。这是因为许多国家不批准这些DNA疫苗，因为用这些疫苗治疗的虹鳟鱼将被标记为转基因生物（GMOs）。因此，设计一个LNP-siRNA系统封装针对g基因的G-siRNA是一个很好的方法来规避这个问题。LNP-G-siRNA将在VHSV挑战的RTGillW1细胞上进行测试，以测试序列特异性敲除并评估对VHSV的保护作用。最终目标是能够彻底研究一种有效的LNP-G-siRNA VHSV治疗方法，并在全球范围内商业化。