mRNA Synthesis and Encapsulation in Ionizable Lipid Nanoparticles.

mRNA vaccines have recently generated significant interest due to their success during the COVID-19 pandemic. Their success is due to advances in mRNA design and encapsulation into ionizable lipid nanoparticles (iLNPs). This has highlighted the potential for the use of mRNA-iLNPs in other settings such as cancer, gene therapy, or vaccines for different infectious diseases. Here, we describe the production of mRNA-iLNPs using commercially available reagents that are suitable for use as vaccines and therapeutics. This article contains detailed protocols for the synthesis of mRNA by in vitro transcription with enzymatic capping and tailing and the encapsulation of the mRNA into iLNPs using the ionizable lipid DLin-MC3-DMA. DLin-MC3-DMA is often used as a benchmark for new formulations and provides an efficient delivery vehicle for screening mRNA design. The protocol also describes how the formulation can be adapted to other lipids. Finally, a stepwise methodology is presented for the characterization and quality control of mRNA-iLNPs, including measuring mRNA concentration and encapsulation efficiency, particle size, and zeta potential. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of mRNA by in vitro transcription and enzymatic capping and tailing Basic Protocol 2: Encapsulation of mRNA into ionizable lipid nanoparticles Alternate Protocol: Small-scale encapsulation of mRNA using preformed vesicles Basic Protocol 3: Characterization and quality control of mRNA ionizable lipid nanoparticles.

mRNA vaccine against malaria tailored for liver-resident memory T cells.

Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions.

Using Full-Spectrum Flow Cytometry to Phenotype Memory T and NKT Cell Subsets with Optimized Tissue-Specific Preparation Protocols.

Full-spectrum flow cytometry is now routinely used in many laboratories internationally, and the demand for this technology is rapidly increasing. With capacity to use larger and more complex staining panels, standardized protocols are required for optimal panel design and analysis. Importantly, for ex vivo analysis, tissue preparation methods also need to be optimized to ensure samples are truly representative of tissues in situ. This is particularly relevant given the recent interest in adaptive immune cells that form residency in specific organs. Here we provide optimized protocols for tissue processing and phenotyping of memory T cells and natural killer T (NKT) cell subsets from liver, lung, spleen, and lymph node using full-spectrum flow cytometry. We provide a 21-color antibody panel for identification of different memory subsets, including tissue-resident memory T (TRM ) cells, which are increasingly regarded as important effectors in adaptive immunity. We show that processing procedures can affect outcomes, with liver TRM cells particularly sensitive to heat, such that accurate evaluation requires fast processing at defined temperatures. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Processing mouse liver for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 2: Processing mouse spleen for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 3: Processing mouse lungs for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 4: Processing mouse lymph nodes for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 5: Staining and flow cytometric analysis of samples for memory T and NKT cell subsets Support Protocol: Obtaining cell counts from flow cytometry data.