Overcoming thermostability challenges in mRNA-lipid nanoparticle systems with piperidine-based ionizable lipids.

Lipid nanoparticles (LNPs) have emerged as promising platforms for efficient in vivo mRNA delivery owing to advancements in ionizable lipids. However, maintaining the thermostability of mRNA/LNP systems remains challenging. While the importance of only a small amount of lipid impurities on mRNA inactivation is clear, a fundamental solution has not yet been proposed. In this study, we investigate an approach to limit the generation of aldehyde impurities that react with mRNA nucleosides through the chemical engineering of lipids. We demonstrated that piperidine-based lipids improve the long-term storage stability of mRNA/LNPs at refrigeration temperature as a liquid formulation. High-performance liquid chromatography analysis and additional lipid synthesis revealed that amine moieties of ionizable lipids play a vital role in limiting reactive aldehyde generation, mRNA-lipid adduct formation, and loss of mRNA function during mRNA/LNP storage. These findings highlight the importance of lipid design and help enhance the shelf-life of mRNA/LNP systems.

Tryptophan nitration of immunoglobulin light chain as a new possible biomarker for atopic dermatitis.

To reduce the incidence and severity of atopic dermatitis, detection and treatment at an early stage are urgently required, but no effective biomarker has been reported. In this study, we attempted to detect a candidate biomarker of early stage atopic dermatitis by focusing on the levels of nitrated residues in the plasma proteins of atopic dermatitis model mice (NC/Nga mice). We found that the immunoglobulin (Ig) light chain was more highly nitrated in the plasma of the animal model than that of control mice. Western blot analysis showed a statistically significant difference between the 6-nitrotryptophan content of the Ig light chain in the NC/Nga mice before onset of atopic dermatitis symptoms and that of the control mice. LC-ESI-MS/MS analysis demonstrated that these light chains contained nitrotryptophan (Trp56) and nitrotyrosine (Tyr66). Immunofluorescence staining revealed a significant increase in manganese superoxide dismutase and inducible nitric oxide synthase production in the skin lesions of the NC/Nga mice. Furthermore, we found protein-bound 6-nitrotryptophan and 3-nitrotyrosine only in the lesioned skin, where their signals partially overlapped with the IgG signal. Our findings suggest that the 6-nitrotryptophan content of Ig light chains could be a new biomarker for detecting early stage atopic dermatitis.

Accumulation of immunoglobulin G against Dermatophagoides farinae tropomyosin in dorsal root ganglia of NC/Nga mice with atopic dermatitis-like symptoms.

Atopic dermatitis (AD), a chronic inflammatory skin disease, manifests as intractable itch, but its underlying mechanisms are poorly understood. This study assessed the relationship between immunoglobulin G (IgG) and dorsal root ganglia (DRG) in NC/Nga mice, a model of AD that manifests AD-like symptoms including itch. Immunohistochemical analysis showed large amounts of IgG in DRG extracts of NC/Nga mice with AD-like dermatitis, with a large fraction of the IgG distributed in satellite glial cells of the DRG. Proteomic analysis showed that this IgG was reactive against tropomyosin of Dermatophagoides farinae. These findings indicate that the accumulation of anti-tropomyosin IgG in DRG of atopic NC/Nga mice may be associated with the pathogenesis of AD-like symptoms, including itch.

Study of normal and pathological blood vessel morphogenesis in Flt1-tdsRed BAC Tg mice.

Blood vessel development and network patterning are controlled by several signaling molecules, including VEGF, FGF, TGF-ß, and Ang-1,2. Among these, the role of VEGF-A signaling in vessel morphogenesis is best understood. The biological activity of VEGF-A depends on its reaction with specific receptors Flt1 and Flk1. Roles of VEGF-A signaling in endothelial cell proliferation, migration, survival, vascular permeability, and induction of tip cell filopodia have been reported. In this study, we have generated Flt1-tdsRed BAC transgenic (Tg) mice to monitor Flt1 gene expression during vascular development. We show that tdsRed fluorescence is observed within blood vessels of adult mice and embryos, indicative of retinal angiogenesis and tumor angiogenesis. Flt1 expression recapitulated by Flt1-tdsRed BAC Tg mice overlapped well with Flk1, while Flt1 was expressed more abundantly in endothelial cells of large blood vessels such as dorsal aorta and presumptive stalk cells in retina, providing a unique model to study blood vessel development.

Genome-wide identification of endothelial cell-enriched genes in the mouse embryo.

The early blood vessels of the embryo and yolk sac in mammals develop by aggregation of de novo-forming angioblasts into a primitive vascular plexus, which then undergoes a complex remodeling process. Angiogenesis is also important for disease progression in the adult. However, the precise molecular mechanism of vascular development remains unclear. It is therefore of great interest to determine which genes are specifically expressed in developing endothelial cells (ECs). Here, we used Flk1-deficient mouse embryos, which lack ECs, to perform a genome-wide survey for genes related to vascular development. We identified 184 genes that are highly enriched in developing ECs. The human orthologs of most of these genes were also expressed in HUVECs, and small interfering RNA knockdown experiments on 22 human orthologs showed that 6 of these genes play a role in tube formation by HUVECs. In addition, we created Arhgef15 knockout and RhoJ knockout mice by a gene-targeting method and found that Arhgef15 and RhoJ were important for neonatal retinal vascularization. Thus, the genes identified in our survey show high expression in ECs; further analysis of these genes should facilitate our understanding of the molecular mechanisms of vascular development in the mouse.