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

本研究项目旨在研制、表征和开发包裹信使RNA(信使RNA)和小干扰RNA(SiRNA)等核酸的脂质纳米颗粒(LNP)系统，用于鱼类健康的基础研究和未来水产养殖疗法的发展。我的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系统以前从未在鱼类细胞上进行研究，我的研究计划将涉及使用体外鱼类细胞培养的深入调查。包裹核酸的LNP是一种令人兴奋的当前治疗选择。2021年8月，FDA批准了LNP-mRNAs治疗，称为辉瑞-生物科技新冠肺炎疫苗(ComirNaty)。此外，2020年12月，FDA发布了现代新冠肺炎疫苗(SPIKEVAX)的紧急使用授权。Comirnaty和SPIKEVAX疫苗已被用于全球数十亿人的联合努力，以结束新冠肺炎大流行。包裹siRNA的荧光标记LNPs将被用于检测各种鱼类细胞的摄取，包括虹鱼(RT)鳃上皮细胞(RTGillW1)、肠道上皮细胞(RTGutGC)、巨噬细胞(RTS11)、嗅觉和肌肉细胞。这些荧光标记的LNPs也将被用于研究鱼类细胞摄取(内吞)的机制。分子抑制剂和荧光摄取标记将被用来确定摄取的确切机制。然后将通过将GFP-siRNA包裹在LNP中来评估基因沉默能力。然后将在稳定表达TurboGFP的RTS11细胞系中检查LNP-GFP-siRNAs的基因沉默能力。此外，还将在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设计一种新的治疗VHSV的方法。VHSV是一种已知的疾病，当被引入水体中时，这种疾病会导致高达50%的死亡率(在彩虹鲑鱼中)。之前已经为VHSV开发了几种疫苗，包括编码糖蛋白(G)基因的质粒DNA疫苗和编码在VHSV挑战细胞中诱导(G)基因序列特异性沉默的shRNA。两种质粒型DNA疗法都取得了成功，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鱼将被贴上转基因生物(GMO)的标签。因此，设计一个针对G基因的G-siRNA的LNP-siRNA系统是解决这一问题的一种很好的方法。LNP-G-siRNA将在VHSV攻击的RTGillW1细胞上进行测试，以测试序列特异性击倒，并评估对VHSV的保护作用。最终目标是能够彻底研究一种可在全球范围内商业化的有效LNP-G-siRNA VHSV治疗方法。

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.