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

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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.