Development of a Ready-to-Use-Type RNA Vaccine Carrier Based on an Intracellular Environment-Responsive Lipid-like Material with Immune-Activating Vitamin E Scaffolds.

Because of its efficient and robust gene transfer capability, messenger RNA (mRNA) has become a promising tool in various research fields. The lipid nanoparticle (LNP) is considered to be a fundamental technology for an mRNA delivery system and has been used extensively for the development of RNA vaccines against SARS-CoV-2. We recently developed ssPalm, an environmentally responsive lipid-like material, as a component of LNP for mRNA delivery. In this study, a self-degradable unit (phenyl ester) that confers high transfection activity and an immune stimulating unit (vitamin E scaffold) for high immune activation were combined to design a material, namely, ssPalmE-Phe-P4C2, for vaccine use. To design a simple and user-friendly form of an RNA vaccine based on this material, a freeze-drying-based preparation method for producing a ready-to-use-type LNP (LNP(RtoU)) was used to prepare the LNPssPalmE-Phe. The optimization of the preparation method and the lipid composition of the LNPssPalmE-Phe(RtoU) revealed that dioleoyl-sn-glycero phosphatidylethanolamine (DOPE) was a suitable helper lipid for achieving a high vaccination activity of the LNPssPalmE-Phe(RtoU). Other findings indicated that to maintain particle properties and vaccination activity, a 40% cholesterol content was necessary. A single administration of the LNPssPalmE-Phe(RtoU) that contained mRNA-encoding Ovalbumin (mOVA-LNPssPalmE-Phe(RtoU)) demonstrated a significant suppression of tumor progression in a tumor-bearing mouse OVA-expressing cell line (E.G7-OVA). In summary, the LNPssPalmE-Phe(RtoU) is an easy-to-handle drug delivery system (DDS) for delivering mRNA antigens in immunotherapy.

Development of an Alcohol Dilution-Lyophilization Method for the Preparation of mRNA-LNPs with Improved Storage Stability.

The lipid nanoparticle (LNP) is one of the promising nanotechnologies for the delivery of RNA molecules, such as small interfering RNA (siRNA) and messenger RNA (mRNA). A series of LNPs that contain an mRNA encoding the antigen protein of SARS-CoV-2 were already approved as RNA vaccines against this infectious disease. Since LNP formulations are generally metastable, their physicochemical properties are expected to shift toward a more stable state during the long-time storage of suspensions. The current mRNA vaccines are supplied in the form of frozen formulations with a cryoprotectant for preventing deterioration. They must be stored in a freezer at temperatures from -80 °C to -15 °C. It is thought that therapeutic applications of this mRNA-LNP technology could be accelerated if a new formulation that permits mRNA-LNPs to be stored under milder conditions were available. We previously reported on a one-pot method for producing siRNA-encapsulated LNPs by combining freeze-drying technology with the conventional alcohol dilution method (referred to herein as the "alcohol dilution-lyophilization method"). In this study, this method was applied to the preparation of mRNA-LNPs to provide a freeze-dried formulation of mRNA LNPs. The resulting formulation can be stored at 4 °C for at least 4 months.

Ready-to-Use-Type Lyophilized Lipid Nanoparticle Formulation for the Postencapsulation of Messenger RNA.

Based on the clinical success of an in vitro transcribed mRNA (IVT-mRNA) that is encapsulated in lipid nanoparticles (mRNA-LNPs), there is a growing demand by researchers to test whether their own biological findings might be applicable for use in mRNA-based therapeutics. However, the equipment and/or know-how required for manufacturing such nanoparticles is often inaccessible. To encourage more innovation in mRNA therapeutics, a simple method for preparing mRNA-LNPs is prerequisite. In this study, we report on a method for encapsulating IVT-mRNA into LNPs by rehydrating a Ready-to-Use empty freeze-dried LNP (LNPs(RtoU)) formulation with IVT-mRNA solution followed by heating. The resulting mRNA-LNPs(RtoU) had a similar intraparticle structure compared to the mRNA-LNPs prepared by conventional microfluidic mixing. In vivo genome editing, a promising application of these types of mRNA-LNPs, was accomplished using the LNPs(RtoU) containing co-encapsulated Cas9-mRNA and a small guide RNA.

Plasmid DNA Mono-Ion Complex for in Vivo Sustainable Gene Expression.

To cleave biocompatible poly(ethylene glycol) (PEG) from the mono-ion complex (MIC) for sustainable cellular uptake in vivo, ω-amide-pentylimidazolium end-modified PEG with an ester bond, that is, APe-Im-E-PEG, has been synthesized. The hydrolysis of the resulting APe-Im-E-PEG proceeded during the incubation for 2 weeks under physiological conditions, which was confirmed by gel filtration chromatography. APe-Im-E-PEG formed the MIC with plasmid DNA (pDNA), assessed by agarose gel retardation assay. Furthermore, dynamic light scattering measurement and transmission electron microscopy observations have estimated that the particle size of the resulting MIC was approximately 30 nm, with a rather flexible structure. The APe-Im-E-PEG/pDNA MIC incubated for 2 weeks exhibited hemolytic activity at endosomal pH, presumably because the pH-sensitive carboxyl groups revealed after the hydrolysis of an ester bond of APe-Im-E-PEG. The APe-Im-E-PEG/pDNA MIC enhanced the gene expression 2 weeks after transfection in vivo by intramuscular administration in mice. Consequently, in vivo sustainable gene expression has been achieved by the molecular design of APe-Im-E-PEG for cellular uptake and endosomal escape proceeded by temporal hydrolysis of the ester bond.