Recently messenger RNA （mRNA） therapeutics is received much attention as one of the vaccination therapies to compete against the coronavirus disease 2019 （COVID-19） pandemic. mRNA therapeutics are generally produced by in vitro transcription utilizing RNA polymerase mediated elongation. However, its purity, stability, and protein synthesis ability, are difficult to be precisely controlled, which is pointed out as drawbacks that must be overcome. To overcome these issues, the introduction of chemically modified nucleic acids is focusing attention. However, it is difficult to flexible molecular design due to the requirement of RNA polymerase recognition ability of chemically modified nucleic acids under in vitro transcription reaction. In the future, the development of a new mRNA design concept based on a flexible molecular design by the progress of chemically modified mRNA therapeutics synthesis method. Under the situation, the authors are focusing on the translation mechanism of mRNA and proposing a new mRNA molecular design to accelerate the translation reaction cycle. In this paper, we introduce an update on therapeutic mRNA design.

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In the case of mRNA drugs, therapeutic effects are caused by proteins produced from mRNAs rather than mRNAs themselves. Thus, a system to regulate therapeutic protein production from mRNAs can function as a kind of drug delivery system. Regulation of protein production from mRNA drugs is more difficult than that from plasmid DNAs or viral vectors as mRNA drugs cannot utilize transcriptional regulatory sequences. However, various translational regulator systems have recently been developed. In this review, we introduce these translational regulator systems including our own studies.

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Recently, with the success of the COVID-19 vaccine, mRNA therapeutics have received a great deal of attention as a next-generation biopharmaceutical. One of the current key issues in mRNA therapeutics is the development of delivery vehicles with higher safety and targetability, excepting the liver. Herein, we introduce a systematic design strategy of cationic polypeptides and their polyplexes for enhanced mRNA delivery. Indeed, a series of cationic polypeptides were synthesized by the aminolysis reaction of poly（β-benzyl-L-aspartate） with varying amine compounds. First, cationic polyaspartamide derivatives were developed for efficient endosomal escape（or endosomal membrane destabilization） by highlighting the acidic pH-sensitivity of aminoethylene（-NHCH₂CH₂-） units. Notably, a polyaspartamide derivative（PAsp（DET）） bearing diethylenetriamine（DET） moieties allowed efficient endosomal escape of polyplexes with lowered cytotoxicity. Second, varying hydrophobic moieties were introduced into the side chains of polyaspartamide derivative with the DET moieties to enhance the stability of mRNA-loaded polyplexes. The results indicated that the derivatives with a hydrophobicity index（or logD）＞–2.4 elicited efficient mRNA transfection in cultured cells. As a result, a polyaspartamide derivative（PAsp（DET/CHE）） with DET and cyclohexylethyl（CHE） moieties achieved the most efficient mRNA transfection without marked cytotoxicity, allowing the topical mRNA delivery in the ventricle via intracranial/intrathecal administration and the systemic mRNA delivery into the lung via intravenous administration. Third, self-catalytic degradation of cationic polyaspartamide derivatives was investigated by slightly changing the spacer length in the side chains. The main-chain degradation was substantially affected by the spacer length；the loss of one methylene spacer resulted in the 5-fold higher degradation rate. A polyaspartamide derivative（PAsp（EDA）） bearing the shorter spacer showed higher mRNA transfection efficiency in cultured cells with reduced cytotoxicity with an increase in pre-incubation time, compared with those bearing the longer spacers. Altogether, it is demonstrated that the polyplex-mediated mRNA delivery can be dramatically improved by fine-tuning the chemical structure of cationic polypeptides.

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Nucleic acid drugs, such as mRNA and siRNA, have recently been attracting attention, and are promising as drugs that will support the next generation of medicine. For RNA, to act specifically at the target site as a medicine, an efficient method of RNA delivery into the target cells is important. In this review, we focus on target-specific RNA delivery using light, which is one of the various targeting strategies. Specifically, we discuss the use of carriers that deliver RNAs into the cytoplasm via photochemical internalization （PCI） or photothermal mechanisms. These carriers facilitate RNA delivery into the cytoplasm only in target cells irradiated with light, allowing RNA drugs to act target-specifically. Although this special issue is focused on mRNA therapeutics, there are very few reports on light-responsive mRNA delivery. In contrast, there are many reports of light-responsive delivery of small RNA, such as siRNA and miRNA. Thus, this review focuses on examples of small-RNA delivery systems that will be diverted to mRNA delivery in the near future. A few examples of light-responsive mRNA delivery are also described.

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RNA molecules such as messenger RNA（mRNA） for protein complementation and small interfering RNA（siRNA） for gene knockdown have been approved as nucleic acid-based therapeutics. These historical successes will accelerate the innovation of RNA-based medication. In these development, Lipid Nanoparticle（LNP） is making a great contribution as a delivery technology in realizing medical care based on these new modalities. Because of the hydrophilicity and anionic charge of the RNA molecules, plasma/endosomal membrane functions as a biological barrier that interrupts their membrane penetration. An ionizable lipid, the main component of the LNPs, can promote the endosomal escape of the nucleic acids by its membrane destabilization activity in response to the acidic pH. The development of RNA delivery systems is now accelerating to realize a large variety of therapeutics against diseases those were hardly cured by conventionally used small molecule compounds. In this section, the development of current ionizable lipids was summarized from the view point of chemical structure of lipid molecules. As a successful example of mRNA-based therapeutics, current understanding of the RNA vaccine was explained from the viewpoint of vaccine efficacy and mechanism of immune activation. Briefly, LNPs have their own immune-stimulative ability and may establish Th2-biased immunity by themselves. When mRNA or other nucleic acids are loaded, the immune profile will probably change depending on the characteristics of the cargo. On the other hand, empty-LNPs and mRNA-LNPs are considered to promote antibody production through the production of IL-6. However, the cells that produce IL-6, the intra-cellular signaling pathway, and the cell subset responsible for antigen presentation are not yet clear. Understanding these immune-stimulative mechanism will be important for the development of safer and more efficient RNA vaccines and/or mRNA-LNPs for the non-vaccine application. Then, recent advances in the organs/cells specific targeting to control the pharmacokinetics of LNPs were summarized. The properties of mRNA-LNPs such as instability at room temperature are considered to hamper the ligand modification. Further investigation is needed to develop an organ targeting LNPs.

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The high efficacy of mRNA COVID-19 vaccine encourages the wider application of mRNA therapeutics. Protein replacement therapy with mRNA therapeutics is a promising alternative approach for administering trophic factor proteins in central nervous system disorders or enzyme replacement therapy in inherited enzyme deficient diseases. Although the concept to deliver the mRNA in vivo as a drug was demonstrated as early as 1990, mRNA instability hindered subsequent research development. The polymer-based carrier, polyplex nanomicelle, is a novel carrier for in vivo mRNA administration. Here, we introduce the researches of in vivo mRNA administration using the nanomicelle carrier to treat animal models, especially focusing on the central nervous system disorders including Alzheimer’s disease, spinal cord injury and ischemic brain disease. We discuss the advantages of mRNA therapeutics and the characteristics of diseases which are highly suitable for mRNA therapeutics.

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For mRNA delivery to the kidney, the choice of administration route and vector is very important due to the structural characteristics of the kidney. Furthermore, it is necessary to devise a combination of physical methods to improve the transduction efficiency. The transfeted cells depend on the delivery methods. Therefore, it may be useful to identify the transfected cells and evaluate their expression distribution in the development of therapeutic strategies. Since there are few reports in the field of mRNA delivery to the kidney, this review describes gene/nucleic acid delivery, focusing on administration routes and vectors, and introduces mRNA delivery to the kidney.

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